Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media

ABSTRACT

A method for isolating and identifying modified para-nitrobenzyl esterases which exhibit improved stability and/or esterase hydrolysis activity toward selected substrates and under selected reaction conditions relative to the unmodified para-nitrobenzyl esterase. The method involves preparing a library of modified para-nitrobenzyl esterase nucleic acid segments (genes) which have nucleotide sequences that differ from the nucleic acid segment which encodes for unmodified para-nitrobenzyl esterase. The library of modified para-nitrobenzyl nucleic acid segments is expressed to provide a plurality of modified enzymes. The clones expressing modified enzymes are then screened to identify which enzymes have improved esterase activity by measuring the ability of the enzymes to hydrolyze the selected substrate under the selected reaction conditions. Specific modified para-nitrobenzyl esterases are disclosed which have improved stability and/or ester hydrolysis activity in aqueous or aqueous-organic media relative to the stability and/or ester hydrolysis activity of unmodified naturally occurring para-nitrobenzyl esterase.

The U.S. Government has certain rights in this invention pursuant toGrant No. DE-FG02-93CH10578 awarded by the Department of Energy andGrant No. N00014-91-J-1397 awarded by the Department of the Navy.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the creation, optimization and use of newenzyme catalysts for deprotection in organic synthesis. Morespecifically, the invention relates to enzymes optimized to removeester-linked para-nitrobenzyl (pNB) protecting groups from carboxylfunctional groups on β-lactam antibiotics and other compounds. Thisinvention also relates to methods by which such enzymes can be alteredand optimized for specific substrates and reaction conditions.

2. Description of Related Art

The publications and other reference materials referred to herein todescribe the background of the invention and to provide additionaldetails regarding its practice are hereby incorporated by reference. Forconvenience, the reference materials are numerically referenced andidentified in the appended bibliography.

Efficient protection and deprotection of functional groups is criticalto successful organic synthesis of polyfunctional molecules. Syntheticschemes often require that a given functional group be protected ordeprotected selectively, under the mildest conditions and in thepresence of functional groups of similar reactivity or other structuresthat are sensitive to acids, bases, oxidation and reduction. Thesesituations represent severe problems for the synthesis of complex,polyfunctional molecules which cannot, or only with great difficulty, besolved using classical chemical tools.

The array of protecting group techniques can be substantially enrichedby the application of enzymes. Enzymes can discriminate stereoisomers aswell as offer the opportunity to carry out highly chemo- andregioselective transformations. The highly selective nature of enzymesmay be exploited advantageously in the manipulation of protecting groupsand in the synthesis of chiral compounds such as drugs and naturalproducts. Furthermore, enzymes function under mild conditions, oftenoperating at or near room temperature and at neutral, weakly acidic orweakly basic pH values. In many cases they combine a high selectivityfor the reactions they catalyze and the structures they recognize with abroad substrate tolerance. Therefore, the application of enzymes can beviable alternatives to classical chemical protection/deprotectionmethods for the introduction and/or removal of suitable protectinggroups (1). The introduction of new enzymes with reactivities andsubstrate tolerances differing from existing enzymes is highlydesirable.

Carboxy groups are often protected by conversion to the benzyl orpara-nitrobenzyl (pNB) esters (2). Benzyl esters are resistant totreatment with reagents such as trifluoroacetic acid, triethylamine,diisopropylethylamine, but are readily removed by hydrogenolysis (over aPd catalyst). Hydrogenolysis is not appropriate, however, for compoundscontaining double bonds, azides, imines, or activated aldehydes, orother functional groups that will be reduced. Benzyl esters can also becleaved using a zinc catalyst under anhydrous conditions, but the extentof hydrolysis is variable and dependent on the conditions (e.g., timeand temperature) of the reaction. The reaction must be carried out underanhydrous conditions, in an organic solvent. Both the organic solventand catalyst can give rise to toxicity or disposal problems forlarge-scale reactions.

Modification by substitution in the aromatic ring can alter thesensitivity of the benzyl group towards deprotection by acidic reagents.PNB esters display increased resistance to acid hydrolysis.

During total synthesis or chemical modification of an antibiotic,several sites on the antibiotic could be adversely affected by thereagents used to carry out any given reaction step. Para-nitrobenzylalcohol (pNB-OH) is commonly used to protect carboxylic acidfunctionalities in cephalosporin-derived antibiotics (U.S. Pat. No.3,725,359 1975!) (3). The pNB ester linkage is stable enough towithstand the various reaction conditions used in subsequent chemicalsteps. After chemical synthesis is completed, deprotection is requiredto return the cephalosporin-pNB ester to its original and activecarboxylic acid form. The chemistry used to deprotect the carboxylicacid involves a catalytic form of zinc in concentrated organic solvent,and on an industrial scale this process generates large amounts ofsolvent and zinc-containing waste material. Cost is associated withprocessing of waste to make it safe for disposal. In 1975, scientists atEli Lilly & Co. interested in pursuing alternative methods ofdeprotection for higher yield and lower disposal costs began a searchfor an esterase capable of performing this deprotection reaction (3).

The enzyme known as para-nitrobenzyl esterase (pNB esterase) wasdiscovered in 1975 by scientists at Eli Lilly & Co., who screened wholecell preparations of numerous bacterial and fungal cultures for thosepossessing catalytic activity toward the hydrolysis of a p-nitrobenzylprotected cephalosporin (3). A Bacillus subtilis culture (NRRL B8079)showed the highest catalytic activity toward two cephalosporin-derivedpNB-protected substrates of all the cultures tested. Although thereaction yield was high, the enzyme activity was not sufficient toconsider for industrial application.

A chromatographically pure solution of pNB esterase was isolated at EliLilly, and its amino acid sequence partially determined. Using thispartial sequence, DNA primers were constructed and used to isolate andsequence the gene for pNB esterase. This gene was cloned into E. coli,where it was over-expressed to produce pNB esterase in large quantities(4). However, partially purified enzyme preparations of "pNB esterase"could not compete with the speed, economy, or the small reaction volumes(due to lack of solubility of substrate in purely aqueous environments)of the zinc-catalyzed deprotection reaction.

The targeted reaction substrates have changed over the fifteen yearperiod as well. Cephalosporin-derived antibiotics continued to evolvefrom the first generation cephalexin (one of the two originalcephalosporin substrates used to search for pNB esterase), secondgeneration cefaclor, third generation cefixime, and fourth generationloracarbef. These antibiotics have been developed to be readily absorbed(generation one), more potent (generation two), much more potent(generation three), and, finally, immensely more stable in the body(generation four) (5). They all are synthesized using the pNB esterprotecting group (6). In protected form, with perhaps the exception ofcefixime, all are only sparingly soluble in water.

The pNB esterase enzyme has been further characterized (6). It is awater soluble, monomeric serine esterase of 54 kD molecular weight and apI of 4.1. The enzyme is active on a variety of ester substrates,ranging from the cephalosporin-derived compounds on which it wasscreened to a number of simple organic esters. Reported KM values forcephalosporin-derived substrates are in 0.5 to 2 mM range. The enzymefunctions best at temperatures below 50° C., and its pH optimum isbetween 8 and 9.

The pNB esterase still suffers from a problem common to a large numberof enzyme reactions in the performance of synthetic chemistry: thedesired substrates are only sparingly soluble in water, and the enzyme'scatalytic ability is drastically reduced by even small quantities ofwater-miscible non-aqueous solvents.

In view of the above problems with the existing pNB esterase, there is acontinuing need to develop new enzymes which have expanded catalyticcapabilities. In particular, new enzymes are needed which can be used toprovide ester cleavage for a variety of substrates and settings,including polar non-aqueous solvents.

SUMMARY OF THE INVENTION

In accordance with the present invention, modified para-nitrobenzylesterases are provided which have improved stability and/or esterhydrolysis activity in aqueous or aqueous-organic media relative to thestability and/or ester hydrolysis activity of unmodified naturallyoccurring para-nitrobenzyl esterase. Unmodified para-nitrobenzylesterase has an amino acid sequence which includes numbered positionsranging sequentially from 1 to 489 (SEQ. ID. NO. 2).

As a feature of the present invention, it was discovered thatsubstitution of amino acids at one or more specific amino acid positionsresulted in the formation of enzymes having improved capabilities inaqueous and aqueous-organic media. The specific amino acid positionnumbers at which substitutions are made to achieve the modifiedpara-nitrobenzyl esterases in accordance with the present invention areposition Nos. 60, 94, 96, 144, 267, 271, 322, 334, 343, 358 and 370.

As a further feature of the present invention, specific amino acidsubstitutions are disclosed which provide specific modifiedpara-nitrobenzyl esterases having improved stability and/or esterhydrolysis activity in organic media. The specific amino acidsubstitutions include I60V, S94G, N96S, L144M, K267R, F271L, H322R,L334V, L334S, A343V, M358V and Y370F. In accordance with the presentinvention, one or more of the specific substitutions increases theenzymatic activity and/or stability of the esterases in aqueous andaqueous-organic media. Ten specific modified para-nitrobenzyl esterasesare disclosed which show enhanced activity in aqueous or aqueous-organicmedia over naturally occurring para-nitrobenzyl esterase. The amino acidsequences for these modified esterases are set forth in SEQ. ID. NOS. 4,6, 8, 10, 12, 14, 16, 18, 20, and 22. These variants are also identifiedthroughout the specification as 1-1h9, 2-19e10, 3-10c4, 4-38b9, 4-43e7,4-54b9, 2-13f3, 2-23e1, 4-53d5 and 5-1a12, respectively. The naturallyoccurring esterase is identified as O-Wtpnb and is set forth in SEQ. ID.NO. 2.

As another feature of the present invention, a method is provided forisolating and identifying modified para-nitrobenzyl esterases whichexhibit improved stability and/or esterase hydrolysis activity towardselected substrates and under selected reaction conditions relative tothe unmodified para-nitrobenzyl esterase. The method involves preparinga library of modified para-nitrobenzyl esterase nucleic acid segments(genes) which have nucleotide sequences that differ from the nucleicacid segment which encodes for unmodified para-nitrobenzyl esterase. Thelibrary of modified para-nitrobenzyl nucleic acid segments is expressedto provide a plurality of modified enzymes. The clones expressingmodified enzymes are then screened to identify which enzymes haveimproved esterase activity by measuring the ability of the enzymes tohydrolyze the selected substrate under the selected reaction conditions.Further modified variants can be produced by accumulating the beneficialmutations identified in this manner.

As an additional feature of the present invention, improvements in thecatalytic activity of modified para-nitrobenzyl esterases with respectto a particular para-nitrobenzyl ester compound is determined byscreening the modified enzymes with a substrate that is thepara-nitrophenyl ester of the compound of interest. For example,screening of esterases for their ability to hydrolyze para-nitrobenzylloracarbef is accomplished by screening the enzymes ability to hydrolyzepara-nitrophenyl loracarbef. The use of para-nitrophenyl ester as ascreening substrate is especially well-suited for screening largenumbers of modified esterases because enzymatic activity is easilymeasured due to the generation of a colored product, i.e.para-nitrophenol. The yellow colored para-nitrophenyl cleavage productis easily measured to provide an accurate measure of the modified estersability to hydrolyze the specific para-nitrobenzyl ester compound.Further, the ability of the modified enzymes to hydrolyzepara-nitrophenyl ester is a good indication of the enzyme's ability tohydrolyze para-nitrobenzyl groups. This method allows the screening oflarge numbers of slightly different variations of enzymes which havebeen produced by random mutagenesis. This ability to easily screen largenumbers of modified enzymes for their esterase activity increases thelikelihood of identifying additional enzymes having increased activityin aqueous or aqueous-organic media and on other related substrates.

The above discussed and many other features and attendant advantages ofthe present invention will become better understood by reference to thefollowing detailed description when taken in conjunction with theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a-c are diagrammatic representations of the substrates andproducts of reactions catalyzed by para-nitrobenzyl esterase andmodifications thereof.

FIG. 1a shows the reaction wherein loracarbef nucleus-para-nitrobenzylester is hydrolyzed.

FIG. 1b shows the reaction wherein para-nitrophenyl acetate ishydrolyzed.

FIG. 1c shows the reaction wherein loracarbef nucleus-para-nitrophenylester is hydrolyzed.

FIG. 2 is a diagram showing the amino acid substitution progression inpara-nitrobenzyl esterase variants. The esterase variants are labeled inbold-type and boxed beneath each label are the amino acid substitutionspresent in each variant. The amino acid substitutions were determined bytranslation of DNA sequencing results.

FIGS. 3a-3o show the DNA sequence alignment of enzyme variantsidentified in accordance with the present invention as they align withnaturally occurring para-nitrobenzyl esterase (O-Wtpnb). The variantsare listed from top to bottom by generation. Boxed regions indicate DNAsequence regions where all variants are identical. The columns of DNAbases not boxed are those where at least one mutation in one of thevariants has occurred.

FIGS. 4a-4e are the amino acid sequence alignment of the enzymevariants. The variants are listed from top to bottom by generation. Theboxed regions indicate amino acid sequence regions where the variantsare all identical with naturally occurring para-nitrobenzyl esterase.The columns of amino acids not boxed are those where at least onemutation in one of the variants has occurred.

FIG. 5a shows variant and wild type pNB reaction kinetics onp-nitrophenyl acetate (pNPA) in 15% dimethylformamide (DMF). Enzymeswere added to a 30° C. reaction solution consisting of 0.1M Tris-HCl pH7.0, 15% DMF, and varying concentrations of pNPA.

FIG. 5b is a plot of variant and wild type pNB esterase reactionkinetics on p-nitrophenyl loracarbef nucleus (LCN-pNP) in 15%dimethylformamide (DMF). Enzymes were added to a 30° C. reactionsolution consisting of 0.1M Tris-HCl pH 7.0, 15% DMF, and varyingconcentrations of LCN-pNP.

FIG. 5c shows graphic results of hydrolysis product formation, asmeasured by HPLC, by variant and wild type pNB esterase on 1.0 mMp-nitrobenzyl loracarbef nucleus (LCN-pNB) in 15% dimethylformamide(DMF). Enzyme were added to a 30° C. reaction solution consisting of 0.1mM Tris-HCl pH 7.0, 15% DMF, and 1.0 mM LCN-pNB.

FIG. 6 shows hydrolysis product formation, as measured by HPLC, ofvariant and wild type pNB esterases on 0.25 mM p-nitrobenzyl loracarbefnucleus (LCN-pNB) in 2.5 and 15% dimethylformamide (DMF) at 30° C. Theresulting peak areas were normalized to that of the parent of generationthree, 2-19E10.

FIG. 7a is a plot of hydrolysis rates of fourth generation variants onthe LCN-pNP and LCN-pNB substrates. The rates are normalized to those ofthe third generation variant 3-10C4. After an 8 hour induction period,whole cell screening assays were performed at 25° C. in a 0.1 mMTris-HCl pH 7.0, 15% dimethylformamide reaction solution containing 0.8mM of either p-nitrophenyl loracarbef nucleus (LCN-pNP) or p-nitrobenzylloracarbef nucleus (LCN-pNB).

FIG. 7b is graphic results of the screening activity of ligation mixture4-38B9 and 4-54B9. The horizontal line indicates the activity of themost active fourth round variant, 4-54B9. Colony 12, which exhibits anapproximate 2-fold improvement in activity, corresponds to pNB esterasevariant 5-1A12.

FIG. 8a is a plot of variant and wild type pNB esterase reactionkinetics on p-nitrophenyl acetate (pNPA) in aqueous buffer (0%dimethylformamide). Enzymes were added to a 30° C. reaction solutionconsisting of 0.1M PIPES pH 7.0 and varying concentrations of pNPA.

FIG. 8b is a plot of variant and wild type pNB esterase reactionkinetics on p-nitrophenyl acetate (pNPA) in 15% dimethylformamide (DMF).Enzymes were added to a 30° C. reaction solution consisting of 0.1MPIPES pH 7.0, 15% DMF, and varying concentrations of pNPA.

FIG. 8c is a plot of variant and wild type pNB esterase reactionkinetics on p-nitrophenyl acetate (pNPA) in 30% dimethylformamide (DMF).Enzyme were added to a 30° C. reaction solution consisting of 0.1M PIPESpH 7.0, 30% DMF, and varying concentrations of pNPA.

FIG. 9a is a plot of variant wild type pNB esterase kinetics onp-nitrophenyl loracarbef nucleus (LCN-pNP) in 1% dimethylformamide(DMF). Enzymes listed were added to a 30° C. reaction solutionconsisting of 0.1M PIPES pH 7.0, 1% DMF, and varying concentrations ofLCN-pNP.

FIG. 9b is a plot of variant and wild type pNB esterase reactionkinetics on p-nitrophenyl loracarbef nucleus (LCN-pNP) in 15%dimethylformamide (DMF). Enzymes were added to a 30° C. reactionsolution consisting of 0.1M PIPES pH 7.0, 15% DMF, and varyingconcentrations of LCN-pNP.

FIG. 9c is a plot of variant and wild type pNB esterase reactionkinetics on p-nitrophenyl loracarbef nucleus (LCN-pNP) in 30%dimethylformamide (DMF). Enzymes were added to a 30° C. reactionsolution consisting of 0.1M PIPES pH 7.0, 30% DMF, and varyingconcentrations of LCN-pNP.

FIG. 10a is a plot of variant and wild type pNB esterase reactionkinetics on p-nitrobenzyl loracarbef nucleus (LCN-pNB) in 1%dimethylformamide (DMF). Enzymes were added to a 30° C. reactionsolution consisting of 0.1M PIPES pH 7.0, 1% DMF, and varyingconcentrations of LCN-pNB.

FIG. 10b is a plot of variant and wild type pNB esterase reactionkinetics on p-nitrobenzyl loracarbef nucleus (LCN-pNB) in 15%dimethylformamide (DMF). Enzyme were added to a 30° C. reaction solutionconsisting of 0.1M PIPES pH 7.0, 15% DMF, and varying concentrations ofLCN-pNB.

FIG. 11 shows the chemical structure of L-glutamine p-nitrobenzyl ester.

FIG. 11a-b shows graphic results of HPLC measurement of the product ofvariant wild type pNB esterase reaction on 1.0 mM L-glutaminep-nitrobenzyl ester in 1% dimethylformamide (DMF) after a fixed reactiontime. Enzyme was added to a 25° C. reaction solution consisting of 0.1MPIPES pH 7.0, 1% (FIG. 11a) or 20% (FIG. 11b) DMF.

FIG. 12 shows graphic results of HPLC measurement of product of variantand wild type pNB esterase reaction on 1.0 mM p-nitrobenzyl benzoate in20% dimethylformamide (DMF). The reaction was performed in 0.1M PIPES pH7.0, 20% DMF and 1.0 mM substrate, 30° C.

FIG. 13 shows the pH-activity profiles from pH 7.0 to 8.25 for a variantfrom each generation. The activity values for each variant arenormalized to its maximal value. The enzyme samples were added to a 30°C. reaction solution consisting of 1% dimethylformamide (DMF), 0.5 mMp-nitrophenyl loracarbef nucleus, and 0.1M PIPES pH 7.0 to 8.25.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, a method of directed evolutionis used to identify and isolate modified enzymes which have improvedcatalytic performance over naturally occurring para-nitrobenzyl (pNB)esterases. This technique relies on being able to screen a large numberof slightly different variations of the enzyme (changes in the aminoacid sequence), and then to accumulate those sequence changes.Generating the variations in a random fashion utilizes randommutagenesis, and effective mutations are accumulated in sequentialgenerations of mutagenesis and screening for the property of interest.The serine protease subtilisin, for example, has been evolved to bealmost five hundred times more active than the naturally occurringenzyme in the presence of 60% dimethylformamide (DMF) (7, 8 and 9). Thepresent invention utilizes a related approach to identify and isolatepNB esterases which exhibit improved activity toward selectedsubstrates, such as loracarbef-p-nitrobenzyl ester, and which exhibitimproved activity under selected reaction conditions, such as in thepresence of polar organic solvents.

The method in accordance with the present invention which is used toidentify and isolate modified esterases having improved activityincludes three basic procedures. The first procedure involves thegeneration of large numbers of randomly mutated esterases. The secondprocedure involves screening the many mutated esterases to determinewhich ones exhibit increased catalytic activity in selected substratesunder specific reaction conditions. The third procedure involvesaccumulating further beneficial mutations in an `evolved` or modifiedesterase. The generation of large numbers of randomly mutated esterasesmay be accomplished by any number of known protocols. The preferredprocedure involves generating a library of modified esterase nucleicacid segments which have nucleotide sequences that differ from thenucleotide sequence of the naturally occurring or unmodified enzymesequence. The library of mutated nucleotide sequences is then expressedin accordance with known methods for producing amino acid sequences. Theprocedures for random nucleic acid mutagenesis and expression of themutated nucleic acids is described in References 7, 8, 9 and in U.S.Pat. No. 5,316,935. Many other methods for random mutagenesis andexpression are known, however, and can be implemented for this purpose.

The screening of the amino acid sequences is accomplished by measuringthe ability of the expressed enzymes to hydrolyze a selected substrateunder selected reaction conditions. While screening can be performeddirectly on the desired substrate, the ease of screening can be greatlyimproved by using the p-nitrophenyl ester of the desired substrate. Forexample, if the compound of interest is loracarbef, then thep-nitrophenyl ester of the loracarbef is substituted for thep-nitrobenzyl ester (see FIG. 1c). If the sequence has the desiredesterase enzyme activity, the para-nitrophenyl group will be cleavedfrom the substrate, as shown in FIG. 1c. The resulting freepara-nitrophenol produces a yellow color in the reaction solution, whichcan be easily measured for both qualitative and quantitative evaluationof the amino acid sequences' performance as an esterase. This screeningprocedure is well-suited for evaluating the performance of modifiedpara-nitrobenzyl esterases. This screening procedure may be used aloneor in combination with the screening on the para-nitrobenzyl ester toprovide confirmation of enzyme activity or to provide a more directmeasurement of the ability of the amino acid sequences to catalyzep-nitrobenzyl ester cleavage for a particular compound. Other suitablenucleus compounds include other β-lactam antibiotics, peptides, peptideantibiotics (16), glycosylated peptides or amino acids (17, 18),peptides (19, 20, 21, 22, 23), natural amino acids (protected both atthe C-terminus and/or acidic side chains) (21, 24, 22), non-naturalamino acids (25), and other synthetic intermediates such as2-aminobenzoate, 2-carbobenzoxyaminobenzoate (26),benzyloxycarbonyl-homoserine andbenzyloxycarbonyl-O-diphenylphosphoryl-homoserine (27).

As employed herein, the term "stability," when used in reference to thestability of para-nitrobenzyl enzymes means the half-life of said enzymewhen exposed to a particular set of reaction conditions, such aselevated temperature and/or organic media. In general, the higher thetemperature to which the enzyme is exposed, the shorter the half-life ofsaid enzyme (i.e., the shorter the enzyme retains its activity).Similarly, the greater levels of organic solvent to which said enzymesare exposed, the shorter the half-life of said enzyme. The phrase"catalytic activity" or simply "activity," means an increase in thek_(cat) or a decrease in the KM for a given substrate, reflected in anincrease in the k_(cat) /_(K) M ratio. The above screening proceduresmay be conducted on a wide variety of substrates and under a widevariety of reaction conditions in order to establish the activity and/orstability of the amino acid sequences in different environments. Forexample, the reaction conditions can be varied from simple aqueoussolutions to those containing varying amounts of organic solvents orother medium components. The amount of organic solvent or other mediumcomponents may be varied to any level. The temperature of the reactioncan be varied in order to isolate variants with improved reaction ratesand/or stabilities at different temperatures. Similarly, the pH of thereaction environment can be varied in order to optimize reaction ratesand/or stabilities at different pH values. The reaction conditions maybe varied widely in order to explore the limits of enzyme activity. Thesubstrate can be varied in order to optimize the amino acid sequencesfor individual substrates or for specific combinations of substrates.

The method of the present invention was used to identify and isolatemodified para-nitrobenzyl esterases which have improved ester hydrolysisactivity toward several para-nitrobenzyl ester substrates in reactionsolutions containing varying amounts of dimethylformamide ranging from 1to 30 percent by volume.

The naturally occurring para-nitrobenzyl esterase has an amino acidsequence which includes numbered position ranging from 1 to 489. Theamino acid sequence for this enzyme is set forth in SEQ. ID. NO. 2 andFIG. 4. The nucleotide sequence which expresses the enzyme is set forthin SEQ. ID. NO. 1 and FIG. 3. In accordance with the present invention,it was discovered that substitution of amino acids at one or more of thepositions numbered 60, 94, 96, 144, 267, 271, 322, 334, 358, and 370resulted in the production of an enzyme which exhibited increasedactivity toward various p-nitrobenzyl ester substrates in purely aqueoussolutions and solutions containing a polar organic solvent, i.e.dimethylformamide. Any number of different amino acids may besubstituted at the various identified positions with a large number ofdifferent combinations being possible where substitutions at one or morepositions is accomplished. The preferred amino acid substitutions areset forth below in Table 1.

                  TABLE 1                                                         ______________________________________                                        Amino Acid Position                                                                            Substitution                                                                            Abbreviation                                       ______________________________________                                         60              Ile→Val                                                                          I60V                                                94              Ser→Gly                                                                          S94G                                                96              Asn→Ser                                                                          N96S                                               144              Leu→Met                                                                          L144M                                              267              Lys→Arg                                                                          K267R                                              271              Phe→Leu                                                                          F271L                                              322              His→Arg                                                                          H322R                                              334              Leu→Val                                                                          L334V                                              334              Leu→Ser                                                                          L334S                                              343              Ala→Val                                                                          A343V                                              358              Met→Val                                                                          M358V                                              370              Tyr→Phe                                                                          Y370F                                              ______________________________________                                    

Preferred modified esterases which contain one or more of thesubstitutions set forth in Table 1 are set forth in FIG. 4 and SEQ. ID.NOS. 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22. The nucleotide sequenceswhich expressed the preferred enzymes is set forth in FIG. 3 and SEQ.ID. NOS. 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23, respectively. Theprocedure for isolating and identifying these preferred enzymes is setforth in the examples below.

During the identification of the improved enzymes, the naturallyoccuring pNB esterase gene was subjected to four rounds of sequentialrandom mutagenesis and screening to increase pNB esterase's catalyticability toward the pNB-protected antibiotic loracarbef. Additionally,genes from positive variants isolated from the final (fourth) round werere-combined by site-specific restriction and ligation in order tocombine the beneficial effects of independent mutations. By increasingthe specificity towards a pNB-containing substrate and by increasing thecatalytic ability in mixtures of water and non-aqueous (organic)solvent, the natural enzyme has been evolved into a number of improvedenzymes which provide industrially useful tools for the deprotection ofpNB esters in the amounts of organic solvents required to solubilizesufficient quantities of non-polar substrates. The above exemplaryrandom mutagenesis and screening in accordance with the presentinvention are set forth in the following examples. For the purposes ofthis description, the reaction conditions will be limited to those whichinclude purely aqueous or mixtures of aqueous and organic media, such asdimethylformamide (DMF). It will be understood by those of ordinaryskill in the art that the method of the present invention hasapplications to screening enzyme activity and/or stability under a widevariety of reaction conditions and is not limited only to screening forenhanced enzyme activity in organic solvents.

EXAMPLES OF PRACTICE Introduction

Loracarbef (LCN) is a cephalosporin-derived antibiotic marketed inmodified form under the trade name LORABID. The production of loracarbefis different from many traditional antibiotics in that it is synthesizedchemically with no microbial fermentation steps. This ensures that theantibiotic is free from any microbially-produced toxins generated duringfermentation. While the functional antibiotic requires a free carboxylicacid moiety, the free carboxylic acid creates problems synthetically.Synthesis of loracarbef has therefore been designed to protect thecarboxylic acid through an ester linkage with pNB alcohol. The pNBesterase enzyme is expected to catalyze the deprotection, that ishydrolyze the pNB ester, toward the end of the chemical synthesis. Thisreaction is shown in FIG. 1a. In addition to protecting the carboxylicacid, the ester-linked pNB group makes the resulting compound virtuallyinsoluble in water.

In designing a method which directs the evolution of an enzyme towardsactivity and specificity on a given substrate, several importantparameters require consideration. One such parameter arises fromexamining the frequency with which enzyme variants with enhancedperformance on the desired substrate arise as a result of randommutagenesis. Nature has demonstrated repeatedly that most variations inan enzyme's amino acid sequence either do not alter the enzyme'sstructure or function or are deleterious. This suggests that a largenumber of variants need to be examined to find a variant more effectiveat performing the desired ester hydrolysis in FIG. 1a. A rapid procedureis required to screen large numbers of enzyme variants. Colorimetricassays are most often optimal in this regard. The reaction in FIG. 1a isproblematic for rapid screening of activity because the absorbancespectra of the reactant and the two products are very similar. Inaddition, the reactant and products do not absorb in the visible region,making the rapid assaying of activity difficult.

Para-nitrophenyl acetate (pNPA) is a general esterase substrate. Theenzyme-catalyzed hydrolysis reaction is shown in FIG. 1b. Use of thepNPA substrate solves the absorbance problem, as the nitrophenol productis yellow while the other reaction components are colorless. The abilityof the alcohol oxygen to form resonance structures which participate inconjugation with the phenyl ring gives rise to the yellow color.Lowering the pH below 6.5 severely shifts the resonance structure awayfrom the conjugation and eliminates the yellow color associated withnitrophenol solutions. The ability to form resonance structures alsomakes nitrophenol an excellent leaving group, as demonstrated by pNPA'sgradual hydrolysis in buffer alone. This spontaneous hydrolysisaccelerates with increasing pH, and at pH values above 8.5 occurs almostimmediately. The pNPA substrate is also membrane permeable. Thissubstrate is hydrolyzed rapidly by whole E. coli cells expressing pNBesterase intracellularly. The same cells, but without the expressionplasmid, do not catalyze the conversion. This substrate is stericallyand chemically different from the LCN-pNB substrate, however, and assuch is not the most preferred choice for directing the evolution of theesterase toward activity on LCN-pNB. It does however, allow foroptimization of enzyme expression in new bacterial hosts, where theincrease in amount of enzyme produced translates into increased activityduring screening.

In accordance with the present invention, in order to optimizegeneration of enzymes with improved activity toward the LCN-pNBsubstrate, a preferred alternate or supplemental substrate is utilizedwhich includes the p-nitrophenyl chromophore from the pNPA substrate andas much of the loracarbef nucleus as possible. This substrate is theLCN-pNP substrate whose structure and reaction are shown in FIG. 1c.This substrate is membrane permeable, obviating the need for cell lysisduring screening. The use of LCN-pNP during screening may result in anenzyme with high hydrolytic activity toward LCN-pNP, but not towardLCN-pNB. The validity of this screening approach was therefore verifiedby comparing the activities of nearly 70 pNB esterase variants on thesetwo substrates.

A second parameter in directed evolution experiments is the choice ofreaction conditions used during screening. The more the screeningconditions differ from the desired reaction conditions, the more likelythat variants found to have a positive, effect in screening will notexhibit improved performance in the desired reaction conditions.Therefore the reaction conditions used for screening should mimic asclosely as possible the ultimate desired reaction conditions(temperature, pH, solvent, substrate concentration, reaction time,etc.).

Screening of Variants

The screen consisted of resuspending individual colonies of bacteria ina small volume of buffer and measuring the turbidity of the bacterialsuspension using a spectrophotometer in order to estimate the cellconcentration in the buffered solution. A small volume of this bacterialsuspension was added to a buffered solution containing a pNP substrate,and the release of product is measured by following the formation ofyellow color. The rate of product appearance was normalized to the cellconcentration by the turbidity measurement. This is indicative of enzymeactivity per bacterium; those colonies which generated higher activityto turbidity ratios were retested. The variant pNB esterases containedwithin the best clones were then purified and tested on the screeningsubstrate (i.e. LCN-pNP) to determine the extent of improvement and onthe desired pNB ester to further determine and confirm that theimprovement applies to the substrate of ultimate interest.

Introduction of Random Mutations

The method of the present invention for directing the evolution of pNBesterase involves making a large library of pNB esterase genes, eachwith a small number of random, or nearly random, alterations in the 1500base pair DNA sequence which codes for the pNB esterase. This collectionof DNA sequences is then placed into E. coli, which translates the DNAsequences into the different amino acid sequences. Because the DNAsequence has been altered slightly, the amino acid sequence of theenzyme may be altered. The LCN-pNP and/or pNPA substrates are then usedto screen out those E. coli that are producing an enzyme which appearsto outperform the original. The best performer is then used to repeatthis sequence of events, in multiple generations, until the desiredperformance goal is achieved. DNA sequence analysis of the improvedenzymes provides identification of the amino acid substitutionsresponsible for the observed activity enhancements.

The number of random alterations introduced in the 1500 base pairsequence (substitution frequency) is a third important design parameterin directed evolution methods. If the frequency of alterations is toohigh, most of the enzymes produced will be inactive. If the frequency ofalterations is too low, most of the DNA base substitutions produced willbe an exact copy of the original DNA sequence, and the resulting enzymeswill not be any different than the original. Because approximatelyone-third of the altered DNA sequences lead to the same amino acidsequence in a protein, the preferred number of DNA alterations per geneis greater than one. At one alteration or less per sequence, much of theDNA produced will produce exact copies of the original protein sequence,and a substantial portion of the screening effort will be spentsearching through copies of the original enzyme. At more than than threealterations per sequence, on average more than two amino acidalterations per enzyme are being produced. The enzyme's activity is afunction of all the alterations contained within; the activity becomes acompetition between the rare alterations which are beneficial and theless rare alterations which are deleterious (7). The preferred number ofalterations is therefore greater than one and not too much larger thanthree.

The substitution frequency is calculated as the number of substitutionsmade in a given sequence divided by the number of possible sites forsubstitution and is usually expressed as a percentage (or fractionalsubstitution). Thus the substitution frequency required to generate oneto three substitutions per gene depends on the sequence length of theDNA coding for the enzyme (or the length of the DNA sequence targetedfor random mutagenesis, if smaller). Polymerase chain reaction (PCR)conditions which generate substitution rates from 0.25 to 20substitutions per 1000 base pairs have been characterized (10, 11 and12).

This above method, as exemplified in the following examples, can be usedto direct the evolution of pNB esterase's ability to better catalyze adesired reaction. Evolution also implies accumulating improvements inactivity over several generations, and this process is repeated multipletimes, each time beginning with the best variant from the previousgeneration. A large library of genes each containing a small number DNAsubstitutions are generated using error-prone PCR techniques. Thislibrary is placed in E. coli, where it is translated from DNA to enzyme.The enzyme library is screened for those enzymes which outperform theoriginal. The best new enzyme then becomes the original as the processis repeated until a desired result is achieved.

Purification of Enzymes

The purification of enzymes was accomplished by using a modification ofthe scheme which includes a pH precipitation, an ammonium sulfatefractionation and three chromatographic steps (6 and 4). The usual threechromatographic steps were reduced to two by replacing a dye affinitycolumn and an ion exchange column with a single (IDA--Cu²⁺) metalaffinity column (IMAC). The wild type pNB esterase open reading framecontains 12 histidines, which are the amino acid residues generallyresponsible for retention on a metal affinity column (13). Although thesurface accessibility of these histidines is unknown, the elution of pNBesterase at 4-5 mM imidazole in an imidazole gradient is consistent withone or two histidine interactions with the chromatographic support (14).

After the enzyme samples were exchanged into Tris buffer, pH 7.0, theenzyme concentrations were measured. As evidenced by SDS-PAGE, thepurity of pNB esterase (estimated to be at least 95%) is not compromisedby replacing the two chromatographic steps with one IMAC column. Inaddition to removing a chromatographic step, this replacement alsoconveniently removed the need for dialysis between columns, as the highsalt content after the first ion exchange column does not affect theperformance of the metal affinity column. Dialysis was performed onlyafter separation on the metal affinity column.

Homology Studies

A homology search of the major protein data bases (Protein InformationResource, Swiss Protein, translated GenBank, and Protein Data Bank)revealed that pNB esterase shares significant homology with a number ofknown esterases. Eleven of the most homologous enzymes, representingseven distinct classes of esterases, were chosen for sequencecomparisons with pNB esterase. These enzymes, their EC classification,the organism from which they were isolated, and their percent identityand similarity to pNB esterase are listed in Table 2. The enzymes wereidentified using a BLAST search of the PDB, PIR, SWISS-PROT, andtranslated GenBank databases. Percent (%) identity and % similarity weredetermined using the BESTFIT tool in the GCG software package.

                                      TABLE 2                                     __________________________________________________________________________    Comparison of amino acid sequence between pNB esterase and esterases          isolated                                                                      from various organisms                                                        Enzyme    Code  Species    % Identity                                                                         % Similarity                                                                        Reference                               __________________________________________________________________________    Acetylcholinesterase                                                                    EC3.1.1.7.                                                                          Torpedo californica                                                                      32.5 53.7  16                                                      Oryctolagus cuniculus                                                                    36.0 58.3  17                                      Butyrylcholinesterase                                                                   EC3.1.1.8.                                                                          Oryctolagus cuniculus                                                                    35.0 56.7  18                                      Carboxylesterase                                                                        EC3.1.1.1.                                                                          Oryctolagus cuniculus                                                                    36.7 57.2  19                                                      Homo sapiens                                                                             37.2 58.6  20                                                      Dictyostelium discoideum                                                                 34.4 55.5  21                                      Thioesterase                                                                            EC3.1.2.14.                                                                         Anas platyrhynchos                                                                       38.7 58.3  22                                      Thriacylglycerol lipase                                                                 EC3.1.1.3.                                                                          Geotrichum candidum                                                                      30.4 48.6  23                                                      Candida rugosa                                                                           29.1 49.5  24                                      Cholesterol esterase                                                                    EC3.1.1.13.                                                                         Candida rugosa                                                                           29.6 49.8  25                                      Carbamate hydrolase                                                                     EC3.1.1.--.                                                                         Anthrobacter oxidans                                                                     34.0 56.8  15                                      __________________________________________________________________________

The cholinesterases are important in neurotransmission, carboxyl- andthioesterases are digestive enzymes, lipases and cholesterol esteraseswork on degrading lipid components. Carbamate hydrolase was discoveredin the same way pNB esterase was discovered: screening for an enzymewith activity on a desired substrate, phenmedipham, an herbicidecarbamate (15). Carbamates are structurally similar to esters,containing a nitrogen linkage not present in esters (R--N--COO--R' vs.R--C--COO--R'), and are known to inhibit esterases. This degradativeactivity was discovered in an Arthrobacter oxidans strain from soilsamples of phenmedipham-treated fields. Starting from enzymes such asthese, the method of the present invention can be used to prepare andisolate groups of modified esterases or carbamate hydrolases which haveimproved activity over other naturally occurring enzymes, such as thoselisted in Table 2.

Some of the esterases in this group of homologous enzymes are noted forthe feature of substrate inhibition at high substrate concentration(26). In particular, substrate inhibition has been a well-noted featureof acetylcholinesterase analysis. While the mechanism of inhibition isnot clear, people have chosen to model the inhibition using the premisethat the substrate can bind at two locations within the enzyme, and doso with different binding constants (27). Butyrylcholinesterase does notshare this inhibition, and this fact is often used to distinguish thetwo cholinesterases. Studies have determined some of the residuesresponsible for this behavior by altering acetylcholinesterase residuesto the appropriate butyrylcholin-esterase residues and examining theinhibitory behavior (28). The fact that the inhibition characteristicscan be altered by substitution shows that enzyme variants of the abovetypes of enzymes, which are not inhibited by substrate, can be producedby the method of the present invention involving random mutagenesis andscreening in high concentrations of substrate for enhanced activity.

Random Mutagenesis of pNB Esterase by Error-Prone PCR

The pNB esterase gene is flanked by the restriction site Xba I 51 basepairs prior to the start of the open reading frame and by therestriction site Bam HI 313 base pairs after the stop codon of the openreading frame (4). Small, single-stranded DNA primers were synthesizedto complement regions 25 base pairs upstream of the Xba I site (forwardprimer) and 143 base pairs downstream of the Bam HI site (reverseprimer). The locations of these primers were chosen because the DNAbetween the two primers is the region that will be altered and amplifiedduring the mutagenic polymerase chain reaction (error-prone PCR). Theerror-prone PCR conditions used were based on the requirements that thesubstitution frequency be between one and three substitutions perthousand bases (1.5 to 4.5 substitutions per gene) (10). Changes in anypart of the open reading frame resulting in enhanced activity areuseful. Therefore the whole open reading frame was given the opportunityto be altered by the mutagenesis. Additionally, once the DNA isamplified and mutagenized, it must be inserted into a circular DNAplasmid. By cutting the amplified DNA with the restriction enzymes Xba Iand Bam HI, the ends of this insert are properly prepared to ligate tothe plasmid. Finally, the primers are located far enough outside of therestriction sites that the small pieces of DNA liberated when the insertis cut by Xba I and Bam HI were visible by standard gel electrophoresistechniques. This ensures that the cutting step has occurred properly,should the ligation perform poorly.

Screening and Analysis of pNB Esterase Variants

An initial round of error prone PCR was performed to producesubstitutions within the pNB esterase gene. The resulting DNA productwas cloned into the expression vector and expressed in E. coli. Onethousand of the resulting colonies were screened for esterase activityon the pNPA substrate (FIG. 1B) in 20% DMF in 96 well plates(experimental details are given in Materials and Methods section below).Of the 1000 colonies selected, thirty-three were rescreened as potentialpositive variants. The three colonies with the highest activity to celldensity (turbidity) ratio were grown, along with the wild type pNBesterase, in 1 liter cultures, and the pNB esterase variants werepartially purified using the precipitation, ammonium sulfatefractionation, and a single DE-52 ion exchange column (6). Thesepartially purified enzymes were then assayed along with wild typecontrol for their hydrolytic activity toward the pNPA and LCN-pNBsubstrates.

All the variants showed higher total activity than wild type pNBesterase on the pNPA substrate, while only one, 1-1H9 (SEQ. ID. NO. 4),showed a significant increase in total activity on the targeted LCN-pNBsubstrate (1H9 indicates the variant designation; the initial1--indicates round or generation number. This should be read "variant1H9 of generation 1.") All variant designations in this specificationfollow this format. This variant was therefore used as the parent forthe second generation of mutagenesis and screening.

The second generation of the directed evolution process began witherror-prone PCR on the gene isolated from variant 1-1H9. 2800 colonieswere screened in 96 well plates, this time using the hybrid LCN-pNPsubstrate and 15% DMF. From these, 65 colonies were rescreened aspotential positive variants, and again the best three were grown in 1liter cultures along with the wild type and the 1-1H9 parent. Themodified pNB esterases (2-13F3--SEQ. ID. NO. 16; 2-19E10--SEQ. ID. NO.6; and 2-23E1--SEQ. ID. NO. 18; 1-1H9--SEQ. ID. NO. 4) and wildtype--SEQ. ID. NO. 2 from these colonies were purified and assayed onall three ester substrates (pNPA, LCN-pNP, and LCN-pNB), with theresults shown in FIGS. 5a, 5b and 5c. While the second round variantshad lost some of their ability to hydrolyze pNPA, all three exhibitedincreased activity on LCN-pNP. For two second round variants, 2-13F3 and2-19E10, the increase in activity also applied to the p-nitrobenzylsubstrate, LCN-pNB. 2-23E1, the variant showing the most activity onLCN-pNP, did not show marked improvement on LCN-pNB. Because 2-19E10showed slightly better performance characteristics on LCN-pNB, it wasused for the third round of mutagenesis.

Screening of the third round of mutagenesis involved examination of 1500colonies using the LCN-pNP substrate and 20% DMF. Forty were rescreenedas potential positive variants. The three best (3-7D5, 3-9E10, and3-10C4--SEQ. ID. NO. 8) were then grown in 500 mL cultures, and theenzymes were purified. Of these three showing best activity on LCN-pNP,only 3-10C4 showed increased activity on LCN-pNB as demonstrated in FIG.6. 3-10C4 shows a 40% improvement in activity over 2-19E10 in 2.5% DMFand a 50% improvement in 15% DMF.

The fourth round of mutagenesis and screening examined 7400 coloniesusing LCN-pNP substrate and 20% DMF. Of these, 250 were rescreened aspotential positives. Sixty-four of those either most active in 5% DMF,most active in 20% DMF, or the best ratio of activities in 20% to 5% DMFwere further screened along with wild type, 1-1H9, 2-19E10, and 3-10C4on LCN-pNB. The screening results on both LCN-pNP and LCN-pNB werenormalized to the activity of the parent 3-10C4 and are shown in FIG.7a. Of the sixty-four colonies chosen, five show activity increases of50% or more over 3-10C4, and sixteen show increases of greater than 20%over 3-10C4. The best five variants were determined based on the abilityto hydrolyze the substrate LCN-pNB only. The best variant, with over a2-fold improvement on 3-10C4, was 4-54B9 (SEQ. ID. NO. 14). Theremaining four variants all demonstrated approximately 60-65%improvement over 3-10C4; these variants were labeled 4-38B9 (SEQ. ID.NO. 10), 4-43E7 (SEQ. ID. NO. 12), 4-53D5 (SEQ. ID. NO. 20) and 4-73B4(later found to be identical to 4-38B9.

A measure of how well the activity of these enzymes on the screening pNPsubstrate relates to activity on the pNB substrate was established (FIG.7a). The overall trend demonstrates a good correlation betweenactivities on the screening pNP and actual pNB substrates, although thedistribution is skewed slightly toward the screening substrate, asdemonstrated by the trend of data points to lie below the forty-fivedegree line. If increases in activity on one substrate correlatedexactly with increases in activity on the other, then all the pointswould lie exactly on the 45-degree line. The strength of thiscorrelation is an important measure of the validity of the screeningstrategy in accordance with the present invention. This graph shows thatthe screening strategy premise that the structurally similar LCN-pNP cansuccessfully replace LCN-pNB, the hydrolysis of which is difficult tomeasure. Contrast this with the pNPA substrate, whose structure does notmimic the desired loracarbef substrate. Modified pNB esterase activitieson pNPA do not correlate as well with activity on the loracarbefsubstrates.

To demonstrate the utility of recombining beneficial mutations in theproduction of improved pNB esterases, a small, biased library of fifthgeneration variants was generated by recombining the genes from thefourth generation variants by restriction and re-ligation. The genesfrom the five variants from the fourth generation were individuallyrestricted by Xho I, a restriction enzyme which cuts in the center ofthis gene. The DNA fragments were mixed with the DNA fragments from4-54B9, the variant which appeared to outperform all others from thefourth generation, in pairwise fashion (e.g. one tube contained thefragments from 4-38B9 and 4-54B9, another tube contained fragments from4-43E7 and 4-54B9, etc.). These mixtures of DNA fragments were eachligated simultaneously with the expression plasmid, transformed into E.coli, and assayed on LCN-pNP substrate in 20% DMF. The results fromscreening colonies expressing the recombined genes are shown in FIG. 7b.Of the four sets of ligations performed, only the mixture ligating4-54B9 with 4-38B9 resulted in an enhancement in activity over 4-54B9.According to the screening data, this combination of mutations (labeled5-1A12--SEQ. ID. NO. 22) displays approximately twice the activity of4-54B9. This demonstrates that positive mutations can be combined foradditional beneficial effects. Further beneficial combinations ofmutations can be found by combining mutations using this or other randomDNA recombination methods or by site-directed mutagenesis, once the DNAsequences are determined.

Kinetic Characterization of Evolved pNB Esterases

Bacteria expressing seven pNB esterases, O-WT (SEQ. ID. NO. 2), 1-1H9,2-19E10, 3-10C4, 4-38B9, 4-43E7, and 4-54B9, were grown in one litercultures, and the enzymes were purified. These purified enzymes wereassayed for their ability to hydrolyze the different ester substrates inthe presence of various concentrations of DMF. FIGS. 8a, b and c showthe specific reaction rates on pNPA as a function of pNPA substrateconcentration for this series of evolved variants from the fourgenerations of mutagenesis and screening. Only 1-1H9 was chosen based onits performance on this substrate, and these assays performed onpurified enzyme suggest that the majority of improvement in activitydemonstrated by this variant during screening is due to an approximatefour-fold increase in amount of enzyme produced. The wild type enzymeoutperforms 1-1H9 in purely aqueous buffer (FIG. 8a). The actualscreening, however, was performed in the presence of DMF, and in 15 and30% DMF, 1-1H9 has higher specific activity towards pNPA than wild type(FIGS. 8b and 8c). Similar trends are seen for the remaining variantsassayed on this substrate. Wild type is the most active enzyme in theabsence of DMF, but is only average among the variants in 15% DMF. In30% DMF, wild type drops still further relative to the pNB esterasevariants. Variants 4-38B9 and 4-43E7, presumably by virtue of havingbeen screened in DMF for four rounds of mutagenesis, are the bestperformers in 30% DMF.

FIGS. 9a, b and c show the results of similar kinetic analyses performedusing the LCN-pNP substrate with which three out of the four rounds ofscreening were carried out. In these plots the results of directedevolution are clearly seen. In 1, 15, and 30% DMF concentrations (FIGS.9a, 9b, and 9c), the two variants least active on this substrate are thewild type enzyme and 1-1H9. This is not surprising given that neither ofthese enzymes had been screened on this substrate. Additionally, as theconcentration of DMF increases, the activity of 1-1H9 increases withrespect to wild type, so that in 30% DMF (FIG. 9c) enzymes displayidentical kinetics. The next enzyme from the bottom is 2-19E10 from thesecond generation of mutagenesis and screening. This variant increasesthe maximum reaction rate by a factor of two over wild type and threeover its parent 1-1H9 at low percentages of DMF. The activity increaseis sensitive to the presence of DMF, decreasing to a smaller improvementin 30% DMF. This trend continues with the 3-10C4 variant, which is 50%faster at producing product than its 2-19E10 parent in 1% DMF. 3-10C4does not lose activity as rapidly as its parent in DMF and is 100%faster than 2-19E10 in 30% DMF. 3-10C4 is the parent of the remainingvariants, all of which show enhanced activity. 4-38B9 shows the leastamount of improvement, with a 20% increase in activity in 1% DMF. Thisincrease in activity is enhanced in DMF to 50% in 30% DMF. 4-43E7 showsa constant two-fold increase in activity across all DMF ranges, and4-54B9 is the most active of all the variants with a constant four-foldincrease in activity over its parent. It is approximately 16 times moreactive than wild type pNB esterase. By comparing the scales of the axes,4-54B9 retains the same activity in 30% DMF as the wild-type enzyme in1% DMF.

FIGS. 10a and b show the kinetic data obtained on the target LCN-pNBsubstrate. All the same trends are observed on the LCN-pNB substratethat were seen on the screening substrate (i.e. the fourth generationvariants are more active than the third generation variant, which ismore active than the second generation variant, etc.), with only minorexceptions. The first exception is that 1-1H9 no longer lags wild typein specific activity on this substrate. The second is that many of thevariants, and especially those in the fourth generation, exhibitslightly lower increases in activity over wild type. For example 4-54B9is now approximately 14 times more active than wild type on LCN-pNB,versus 16 times wild type on LCN-pNP. Additionally, DMF has a biggernegative effect on the LCN-pNB hydrolysis reaction than it does thehydrolysis of LCN-pNP. On LCN-pNP, 15% DMF reduces the maximal activityby a factor of two in the two best variants in 1% DMF, while 15% DMFaffects the LCN-pNB hydrolysis by reducing the maximal activity by afactor of three over the 1% DMF activity.

The results shown in the FIGS. were used to calculate the k_(cat), KMand k_(cat) /KM values reported in Table 3. In the case of the pNPAsubstrate, only k_(cat) /KM is reported because the solubility of thepNPA substrate did not permit the high substrate concentrations requiredto accurately determine the individual k_(cat) and KM parameters. DMFdramatically increases K_(M) while also decreasing k_(cat). For the LCNsubstrates, the mutations accumulated during directed evolution mitigatethe increased K_(M). The effects on k_(cat), however, are moreprominent: k_(cat) increases more than 9-fold from wild type to 4-54B9in 15% DMF, while K_(M) decreases by less than a factor of two onLCN-pNB. This result reflects the relatively high substrateconcentration used during screening (0.8 mM). At substrateconcentrations on the order of K_(M), increased specific activity willresult mainly from improvements in k_(cat). These improvements areobviously the most useful for enzymes intended to be used fortransformations in high substrate concentrations.

                                      TABLE 3                                     __________________________________________________________________________             p-nitrophenyl acetate                                                         0% DMF    15% DMF  30% DMF                                           Variants kcat/Km   kcat/Km  kcat/Km                                           __________________________________________________________________________    0-WT     5.67      0.94     0.24                                              1-1H9    4.05      1.08     0.32                                              2-19E10  3.20      0.89     0.28                                              3-10C4   2.50      0.86     0.33                                              4-38B9   3.04      1.03     0.36                                              4-43E7   2.42      0.81     0.29                                              4-54B9   2.57      0.64     0.27                                              __________________________________________________________________________    p-nitrophenyl loracarbef nucleus                                                      0%        15%        30%                                                      DMF kcat/ DMF kcat/  DMF kcat/                                             kcat                                                                             Km  Km kcat                                                                             Km  Km kcat                                                                              Km  Km                                           __________________________________________________________________________    0-WT 0.14                                                                             0.07                                                                              2.11                                                                             0.10                                                                             2.36                                                                              0.04                                                                             0.024                                                                             7.55                                                                              0.003                                        1-1H9                                                                              0.08                                                                             0.03                                                                              2.47                                                                             0.07                                                                             1.84                                                                              0.04                                                                             0.021                                                                             6.05                                                                              0.003                                        2-19E10                                                                            0.25                                                                             0.09                                                                              2.68                                                                             0.12                                                                             1.50                                                                              0.08                                                                             0.023                                                                             4.67                                                                              0.005                                        3-10C4                                                                             0.35                                                                             0.11                                                                              3.15                                                                             0.21                                                                             1.77                                                                              0.12                                                                             0.044                                                                             5.18                                                                              0.009                                        4-38B9                                                                             0.39                                                                             0.06                                                                              6.18                                                                             0.25                                                                             1.41                                                                              0.17                                                                             0.067                                                                             5.68                                                                              0.012                                        4-43E7                                                                             0.67                                                                             0.08                                                                              8.51                                                                             0.40                                                                             1.27                                                                              0.31                                                                             0.094                                                                             5.09                                                                              0.018                                        4-54B9                                                                             1.44                                                                             0.12                                                                              12.09                                                                            0.93                                                                             1.27                                                                              0.73                                                                             0.141                                                                             2.92                                                                              0.048                                        __________________________________________________________________________           p-nitrobenzyl loracarbef nucleus                                                   0%             15%                                                            DMF  kcat/     DMF  kcat/                                                kcat Km   Km   kcat Km   Km                                            __________________________________________________________________________    0-WT   1.64 0.04 38.27                                                                              0.54 0.88 0.61                                          1-1H9  1.94 0.04 50.75                                                                              0.69 0.85 0.82                                          2-19E10                                                                              4.59 0.04 102.59                                                                             1.51 0.87 2.09                                          3-10C4 6.53 0.07 96.37                                                                              1.62 0.40 4.05                                          4-38B9 8.51 0.04 222.47                                                                             1.89 0.31 6.17                                          4-43E7 9.13 0.03 271.06                                                                             3.02 0.37 8.11                                          4-54B9 18.96                                                                              0.05 396.41                                                                             6.84 0.46 14.74                                         __________________________________________________________________________

Performance of Evolved Enzymes on Other Substrates

To determine the extent to which the newly-evolved enzymes exhibitedincreased general p-nitrobenzyl esterase activity, four purified pNBesterases (0-WT, 1-1H9, 2-19E10, and 4-54B9) were assayed on L-glutaminep-nitrobenzyl ester and p-nitrobenzyl benzoate by HPLC, as described inMaterials and Methods. The resulting product peak areas are shown forthe different substrate/enzyme combinations in FIGS. 11 and 12.L-glutamine pNB is soluble in aqueous buffer. Its enzyme-catalyzeddeprotection in the absence of DMF is shown in FIG. 11a. All the pNBesterases tested catalyze this reaction, with wild type being the mostactive. The activity of the enzyme variant decreases from generation togeneration. The results are altered significantly, however, when 20% DMFis added (FIG. 11b). Under these conditions, 2-19E10 pNB esterasedemonstrates the mosst activity toward removing the pNB-protecting groupfrom L-glutamine p-nitrobenzyl ester. The first generation variant 1-1H9outperforms wild type by 20%, and the second generation variant 2-19E10outperforms the wild type enzyme by 60%. The fourth generation variant4-54B9 apparently introduces a substitution which disrupts the earliergenerations' enhancements of activity: this enzyme has lost the abilityto catalyze this reaction better than wild type.

The hydrolysis of p-nitrobenzyl benzoate, reported in FIG. 12, shows thesame trends demonstrated in FIG. 9b, although more dramatically. Becausep-nitrobenzyl benzoate is not soluble in aqueous buffer, the assays onthis substrate were performed only in 20% DMF. In this environment,1-1H9 is 60% better than wild type, and 2-19E10 is 2.5 times better thanwild type. 4-54B9 again has lost the ability to outperform previousgeneration variants, including the wild type pNB esterase.

pH Studies

The pH optimum for activity of wild-type pNB esterase is 8.3 (6), whilethe screening for evolved pNB esterases was carried out at pH 7.0. Todetermine to what extent the pH-activity profiles of the pNB esterasewere altered, or may have drifted, as a result of directed evolution atpH 7.0, the enzymes' abilities to hydrolyze the LCN-pNP screeningsubstrate was measured as a function of pH. The activities normalized tothe maximum activity for enzyme variants from each of the fourgenerations are shown in FIG. 13. While the pH optima of the enzymevariants have not changed significantly, the pH-activity profiles ofthose from later generations have broadened slightly. In other words,reaction rates at lower pH values increase slightly more during directedevolution than does the maximum rate ˜pH 8.25, which probably once againreflects the choice of pH 7.0 for screening.

Sequence Analysis

FIG. 3 presents the aligned DNA sequences of all the variants sequencedduring this study. The variants are listed in order by generation, andthe sequences start with DNA base one (A of the first codon ATG). TheDNA bases conserved between all members of this pNB esterase family areboxed. Where a mutation has occurred, the column of DNA bases is notboxed, and a dash is indicated in the consensus sequence at the bottomof each set of rows.

Table 4 summarizes the positions of the DNA base changes in the enzymevariants with respect to the wild type pNB esterase gene sequence forall the variants sequenced during this study. Bold type indicates thesubstitutions not present in the previous generation parent enzyme.Horizontal lines indicate the beginning and end of the open readingframe, which starts at base position 1 and ends at position 1470. Alltogether the sequences contain 31 substitutions, of which 29 areunambiguously unique. The two substitutions which may not be unique arethose where identical substitutions were found in two different variantsof the same generation, those being the A to G substitutions at position1075 in two variants from generation 2, and the A to G substitution atposition 181 in two variants of generation 4.

                                      TABLE 4                                     __________________________________________________________________________    DNA    Generation 2                                                                              Gen 3                                                                             Generation 4        Gen 5                              Pos.                                                                             Gen 1                                                                             13F3                                                                              19E10                                                                             23E1                                                                              10C4                                                                              38B9                                                                              43E7                                                                              53D5                                                                              54B9                                                                              73B4                                                                              1A12                               -12                                                                              1H9 A→T  A→T                                                                        A→T                                                                        A→T                                                                        A→T                                                                        A→T                                                                        A→T                                                                        A→T                         __________________________________________________________________________     87                                                                              C→T                                                                        C→T                                                                        C→T                                                                        C→T                                                                        C→T                                                                        C→T                                                                        C→T                                                                        C→T                                                                        C→T                                                                        C→T                                                                        C→T                          102                               T?C                                         181                   A→G      A→G                                                                        A→G                          255                           T→C                                      283                       A→G                                          290   A→G                                                              291               T→C                                                  333       T→A                                                                            T→A                                                                        T→A                                                                        T→A                                                                        T→A                                                                        T→A                                                                        T→A                                                                        T→A                          399           T→C                                                      433               T→A                                                                        T→A                                                                        T→A                                                                        T→A                                                                        T→A                                                                        T→A                                                                        T→A                          720                           T→C                                      803                               A→G                                  814           T→C                                                      968                                                                             A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                         1003                       T→G                                         1004                               T→C                                                                            T→C                         1031           C→T      C→T                                     1075   A→G                                                                        A→G                                                                            A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                         1112                                                                             A→T                                                                        A→T                                                                        A→T                                                                        A→T                                                                        A→T                                                                        A→T                                                                        A→T                                                                        A→T                                                                        A→T                                                                        A→T                                                                        A→T                         1122   A→G                                                             1239       A→G                                                                            A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                         1302   A→G                                                             1485                                                                             A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                         1568                                   T→A                             1618           T→C                                                     1658                                                                             A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                                                                        A→G                         1678                                                                             T→C                                                                        T→C                                                                        T→C                                                                        T→C                                                                        T→C                                                                        T→C                                                                        T→C                                                                        T→C                                                                        T→C                                                                        T→C                                                                        T→C                         1745       C→T                                                                            C→T                                                                        C→T                                                                        C→T                                                                        C→T                                                                        C→T                                                                        C→T                                                                        C→T                         __________________________________________________________________________

FIG. 4 presents the amino acid sequence alignment of the pNB esterasefamily as translated from the DNA sequence alignment in FIG. 3. Asbefore, the variants are listed in order by generation. The sequencesstart with amino acid one, and the DNA bases conserved between allmembers of this pNB esterase family are boxed. Where a mutation hasoccurred, the column of amino acid residues is not boxed, and a dash isindicated in the consensus sequence at the bottom of each set of rows.

FIG. 2 is an "evolution tree" summarizing the amino acid substitutionsand positions resulting from the amino acid sequence information in FIG.4 and Table 4. All three clones sequenced from the second generationcontain the substitutions observed in the 1-1H9 sequence, as well as afew new additional substitutions. This can be seen at the DNA level inTable 4 and at the protein level in FIG. 2. Two of the second generationclones contain the same DNA substitution, an A to G substitution atposition 1075, which gives rise to the substitution of methionine atposition 358 by valine. This residue is believed to be responsible forthe increased specific activity of these two clones, as it is the onlynon-silent mutation in clone 2-19E10, whose catalytic activity isslightly better than 2-13F3. 2-19E10 was chosen to parent the thirdround of mutagenesis. One silent and one translated substitution areadded to the evolutionary sequence in 3-10C4; a T to A substitution atposition 433 gives rise to leucine 144 substituted by methionine. Thenext five sequences are progeny of 3-10C4 from the fourth generation.All have the eight DNA base substitutions of 3-10C4 in common as thecumulative result of the three previous rounds of evolution. 4-38B9 and4-73B4 each contain only an identical open reading frame substitution,an A to G change at position 181, leading to isoleucine at position 60changed to valine. As a result, these two variants are listed togetherin FIG. 2. 4-43E7 and 4-54B9 each contain two substitutions; one occurswithin identical codons in the DNA sequence, giving rise to changes inleucine 334 to valine in 4-43E7 and serine in 4-54B9. 5-1A12 was createdas a combination of 4-38B9 and 4-54B9 by cutting and religating thesetwo genes near the 1000 DNA base position. S-1A12 is therefore expectedto contain the isoleucine 60 to valine substitution from 4-38B9 and theleucine 334 to serine substitution from 4-54B9.

The location versus frequency of substitution was examined and thelocations of substitutions resulting in amino acid changes appearsdistinctly non-random. Fully half of the translated substitutions DNAmutations lie within a 144 base pair stretch of DNA (less than 10% ofthe open reading frame). This is due to the fact that a non-randomselection of enzymes were chosen for sequencing (only those exhibitingimproved activity). This region of the amino acid sequence plays animportant role in substrate recognition and enzyme activity.

Random Mutagenesis

The frequency of substitutions as a function of position within the genesequence was determined. It was found that the locations of DNAsubstitutions in the sequenced genes are well distributed throughout thetarget sequence. Thus the error-prone PCR technique generates variationsat random locations. The types of substitutions generated, however, arenot random. Of the 29 unique substitutions, 25 were substitutionschanging an A or T, 4 were substitutions of C, and none weresubstitutions of G. These bases were changed almost half of the time toG, with A to G changes making up the majority (9/10) of thesesubstitutions. This non-randomness is also shown in the number oftransition (purine to purine changes--A to G or C to T) to transversion(purine to pyrimidine changes--A to T/G or C to A/G or T to A/G)substitutions, where the transitions outnumber the transversions 24 to5.

The conditions used in these examples (differing from normal PCRconditions by increases in all four dNTP concentrations) give a reportederror-rate of between 1.5 and 4 base substitutions per 1000 bases. Underthese conditions, the substitutions should be predominantly transitions(e.g. A to G), no transversions (e.g. A to T), and a small fraction ofinsertion or deletions. The sequence data presented here demonstrate asignificant bias towards A to G substitutions, especially in the firsttwo generations. We found that the substitution frequency was at thehigher limit of the expected range, with the 1-1H9 variant demonstratinga substitution rate of 3.5 substitutions per 1000 bases, and highlypopulated with A to G changes. The third and fourth PCR reactionsyielded lower mutation frequencies, with only 1 to 2.5 substitutions per1000 bases. Transition substitutions still outnumbered transversionsubstitutions by 3 to 1. In no case was a G mutated.

Substitution bias in the resulting protein sequences also arises fromthe fact that the DNA sequence is translated to the amino acid sequencethrough the triplet coding ribosomes. Twenty amino acids are encoded bysixty-one triplet DNA codons, the distribution of these codons is farfrom even. For example, tryptophan is coded for by only one of the 64triplet codons (TGG), while Leu is encoded by six. Single base changeswithin a codon are the only type of base changes we can expect to see,as the probability of making two random substitutions within one codonis small. On average, only five to six new amino acids are available toreplace each amino acid in the original sequence by single basesubstitution. Thus, the translation process introduces non-randomness inthe amino acid sequence. This is useful for directed evolution inaccordance with the present invention, when one does not want toradically alter an enzyme that has some catalytic activity, but ratherwants to incorporate small changes which enhance the enzyme's ability toperform. This bias towards conservative amino acid substitutions isdemonstrated in the examples with most changes being conservative ones,such as tyrosine 370 to phenylalanine in 1-1H9, phenylalanine 271 toleucine in 2-23E1, and isoleucine 60 to valine in 4-38B9.

MATERIALS AND METHODS DNA

The plasmid pNB106R was provided by Eli Lilly & Co (Indianapolis, Ind.).This plasmid contains the pNB esterase gene under the control of analtered 1 promoter, pL106 (U.S. patent application Ser. No. 07,739,280)(4). The plasmid also contains a temperature sensitive 1 CI repressorwhich inactivates the pL106 promoter below 35° C. Further, the plasmidcontains an E. coli origin of replication, a plasmid copy control gene,and a tetracycline resistance gene.

Computer Work

Homology searches and sequence alignments were performed at theCalifornia Institute of Technology's Sequence Analysis Facility usingthe GCG Sequence Analysis Software Package version 8.0 from the GeneticsComputer Group (University Research Park, Madison, Wis.). BLAST searchesof the Brookhaven Protein Data Bank (PDB), the SWISS-PROT database, theProtein Information Resource database, and the translated GenBankdatabase were performed at the National Center for BiotechnologyInformation using a BLAST network service (29). PHYLIP was used at thesame facility to construct evolutionary trees (30).

Restriction of DNA

Plasmid and fragment DNA when required were cut with Bam HI and Xba I(Boerhinger Mannheim, Germany) in restriction buffer B at 37° C. for onehour. The resulting linear DNA was then run on a 1% agarose gel andseparated into bands according to size. The appropriately sized band wasexcised from the gel and extracted using either the GeneClean (Bio101,Vista, Calif.) or Qiagen (Chatsworth, Calif.) method. In both casespurified DNA was eluted in Tris-EDTA buffer.

Random Mutagenesis

The pNB esterase gene (1470 base pairs (bp)) in pNB106R is flanked by anXba I restriction site 51 bp before the start of the ORF and by a Bam HIsite 313 bp downstream from the stop codon (4). PCR primers(3'-GAGCACATCAGATCTATTAAC-5' and 3'-GGAGTGGCTCACAGTCGGT-GG-5') weresynthesized to complement regions 25 bp upstream of the Xba I site and143 bp downstream of the Bam HI site to allow andom mutagenesis over a2000 bp region including the entire pNB esterase open reading frame. Asolution containing 1 mM dNTPs, 16.6 mM (NH₄)₂ SO₄, 67 mM Tris-HCl (pH8.8), 6.1 mM MgCl₂, 6.7 mM EDTA (pH 8.0), and 10 mM b-mercaptoethanol, 6mg of forward and reverse primers, 10 ng of plasmid pNB106R and 2.5units of Taq DNA polymerase (Perkin Elmer-Cetus, Foster City, Calif.) ina total volume of 100 mL were covered with 2-3 drops of light mineraloil (Sigma, St. Louis, Mo.)). The sample was then placed in a wellcontaining 2-3 drops of mineral oil of a Precision Scientific thermalcycler. The thermal cycler repeats the following steps: 1 minute at 94°C., 2 minutes at 42° C., and 1 minute at 72° C. for 25 cycles. Theseconditions should generate an error frequency of approximately onesubstitution per 1000 bases, or approximately 1.5 substitutions per genecopy (10). The fragment of DNA amplified by this technique was thensubjected to a phenol/chloroform extraction and ethanol precipitation.The DNA was restricted and purified as described above.

Competent Cell Preparation

Competent TG1 cells were prepared according to the CaCl2 method (31).TG1 cells were grown overnight at 37° C. in a 3 mL culture of LB broth.The cells were diluted 1:200 in fresh LB and allowed to grow to an OD₆₀₀of 0.35 to 0.40. They were placed on ice for 1 hour and spun at maximumspeed in a 4° C. Beckman tabletop centrifuge. The cell pellet wasresuspended in 0.5 volumes of 0.1M CaCl₂ and allowed to sit on ice for30 minutes to 1 hour and recentrifuged as before. The cell pellets wereresuspended in sterile 0.02 volumes of 0.1M CaCl₂, 10% v/v glycerol andfrozen at -70° C. until use.

Ligation and Transformation

Ligation reactions were performed using T4 DNA ligase(Boerhinger-Mannheim, Germany). Vector DNA (the entire pNB106R plasmidexcluding the pNB esterase gene between Xba I and Bam HI), insert DNA(the pNB esterase gene between Xba I and Bam HI), 10X ligation buffer,water and enzyme were combined and incubated at 4° C. overnight (12-16hours). The solution was incubated with previously prepared competentcells on ice for 1 hour. The cells were then heat shocked at 42° C. for1 minute, supplied with an equal volume of LB media, and incubated at30° C. for 45 minutes. This solution was then plated onto LB platescontaining tetracycline to 20 mg/mL (LB Tet plates).

Screening

Transformants arising from ligations of pNBE vector and randomlymutagenized inserts were allowed to grow for 36 to 48 hours beforeshifting to 42° C. to induce expression of the pNB esterase gene. Afteran eight hour induction period, each colony was picked with a steriletoothpick and resuspended in a unique well of a 96 well plate containing200 mL of 0.1M Tris-HCl, pH 7.0. The turbidity of each well was measuredas the absorbance at 620 nm adjusted by a cell-free reference well by a96 well plate reader. A 20 mL aliquot from each well was pipetted into asecond 96 well plate, to which was added 200 mL of a substrate solutioncontaining 0.8 mM para-nitrophenyl acetate (pNPA) and 0.4% (v/v)acetonitrile or para-nitrophenyl loracarbef nucleus (LCN-pNP), 0.1MTris-HCl pH 7.0, and between 0 and 30% v/v dimethylformamide (EM ScienceGuaranteed Reagent grade). The resulting reaction was monitored usingthe 96 well plate reader at 405 nm. Reactions were typically monitoredfor 11 data points varying from 15 seconds between data points for 0%DMF measurements to 180 seconds between data points for 30% DMF. Theslopes of the best-fit lines through the resulting 11 data points foreach of the 96 wells were normalized by the corresponding absorbance at620 nm measured previously. These normalized values were compared, andthe wells exhibiting the highest activity to turbidity ratios wereplated onto LB Tet plates. Two single colonies from these plates wererestreaked on LB Tet plates to provide single colony isolates forfurther testing. Two single colonies from each of these second plates(four colonies total) were then arrayed onto LB Tet plates using steriletoothpicks. This collection of potential variants was then rescreenedusing the activity to turbidity ratio assay again. Of those that showedbetter activity to turbidity than wild type, the best three were chosenfor larger scale culture and purification.

Crude screening was performed on the fourth generation variants usingthe LCN-pNB substrate using a similar whole cell assay. 0.10 ml samplesof the resuspended colonies were removed from each well of the 96 wellplate and added to a quartz cuvette containing a 1000 ml reactionsolution consisting of 2.5% DMF, 0.1M Tris-HCl pH 7.0, and 0.25 mMLCN-pNB. The absorbance at 291 nm of each sample was measured for 2.5min. using a UV spectrophotometer. Initial rates were measured for boththe LCN-pNP substrate in the 96 well plate assay (above) and the LCN-pNBsubstrate in quartz cuvettes. Both sets of slopes generated from theinitial rate data were normalized to the turbidity measurements at 620nm.

Cell Culture

Single colonies were inoculated into 5 mL LB Tet culture tubes andallowed to grow overnight at 30° C. The contents of these tubes werethen used to inoculate a one-liter culture of LB Tet and allowed to growto maximum turbidity. These one-liter cultures were decanted intosterile centrifuge bottles and spun at 6000 rpms in a JA-10 rotor for 15minutes in a Beckman centrifuge. The cell pellets were resuspended in LBTet pre-warmed to 42° C. The flasks were placed in a 42° C. incubatorand allowed to shake for 8 hours (4). The cells were harvested bysimilar centrifugation and resuspended in a centrifuge tube in 25 mL ofBuffer A (Lysis Buffer), consisting of 10 mM potassium phosphate, 1 mMb-mercaptoethanol, and 0.5 mM EDTA, pH 7.0.

Cell Lysis

A French Press was used to lyse the harvested cells. The lysis wasaccomplished by placing the chilled sample into a steel housing, whichwas compressed to 20,000 atmospheres. A small needle valve was thenopened and the cells were released to ambient conditions, causing thecells to rupture. This process was repeated three times to insurecomplete lysis. The steel housing was kept chilled prior to use at 4° C.and the samples were stored before and after on ice.

Purification

After lysis the cell debris was pelleted by centrifugation at 12,000 gin a JA-20 rotor for 15 minutes at 4° C. (6). The cell lysatesupernatant was adjusted to pH 5.0 with HCl, and the newly formedprecipitate was removed by centrifugation at 12,000 g in a Beckman JA-20rotor for 30 minutes at 4° C. The supernatant volume was measured andammonium sulfate was dissolved to 45% saturation at 0° C. For reference,the ammonium sulfate saturation amount used for calculations was 41.22g/100 mL solution at 0° C. The solution was chilled to 0° C. on ice for5 minutes and centrifuged in a JA-20 rotor at 12,000 g for 30 min. at 4°C. The supernatant was transferred to a new centrifuge tube, whereammonium sulfate was added to bring the final amount to 85% saturationat 0° C. Centrifugation was performed as before, and the supernatantdiscarded. The pellet was redissolved in Buffer B (10 mM Tris-HCl, 50 mMNaCl, 1 mM b-mercaptoethanol, and 0.5 mM EDTA, pH 8.5), placed in anAmicon® spin filtration unit (Centricon--10) and buffer exchanged threetimes with Buffer B to remove the ammonium sulfate. The resultingprotein sample was applied to a DEAE-sepharose column (2.5 cm ID×10 cmhigh) pre-equilibrated in Buffer B. The column was rinsed with buffer Buntil the baseline was restored. The column was then rinsed with bufferC (10 mM Tris-HCl, 50 mM NaCl, pH 7.0) until the pH reached 7.0. An NaClgradient from 50 to 500 mM in buffer C (300 mls total volume) was passedthrough the column and fractions collected. Those fractions containingactivity were pooled and then applied to an immobilized metal affinitychromatography (IMAC) column (2.5 cm ID×10 cm high, Fast-flow ChelatingSepharose, Pharmacia, Sweden) prepared as per the manufacturer'sinstructions. The column was first pre-treated by rinsing with threecolumn volumes of (one column volume was approximately 50 ml) 0.5M NaCl,50 mM EDTA pH 8.5 to remove all chelated metal ions, 2M NaCl to removeany ionically bound material, and 1M NaOH to remove any denaturedprotein. Copper as 100 mM copper sulfate in 100 mM sodium acetate pH 4.6was reloaded onto the IMAC column, washed with 20 mM sodium phosphate,0.5M NaCl, 50 mM imidazole pH 7.2 until pH 7.2, and finally equilibratedwith five column volumes of 20 mM sodium phosphate, 0.5M NaCl, 1 mMimidazole for loading. The sample was applied to the column and thecolumn washed with the 1 mM imidazole solution until baseline wasrestored. A linear gradient formed by 100 mls of 1 mM and 10 mMimidazole solutions (200 mls total volume) was applied, and fractionswere collected. All tubes demonstrating higher than background activitywere pooled, concentrated, and buffer exchanged into 0.1M Tris-HCl, pH7.0 in the Amicon Centricon-10 units as before.

SDS-Page Gels

SDS-Page gels were used to determine purity of protein solutions.Separating gels were made of 10% acrylamide and allowed to gel underbutanol. After gelling the butanol was removed and a 4% acrylamidestacking gel was poured on top of the separating gel. Up to 5 mL ofconcentrated protein samples were mixed with 20 mL loading buffer (10%glycerol, 1% SDS, etc.) and boiled for 4 minutes. The 25 mL samples wereloaded onto the gel and run at 200 V for approximately 30 minutes, atwhich time the loading buffer dye reached the bottom of the gel. The gelwas removed from the apparatus and stained using a Coomasie blue stainsolution. After staining a minimum of 45 minutes, the Coomasie bluestain was poured off and destain was added. This was allowed to incubateuntil the solution approximated the color of the gel, at which time thedestain was poured off and new destain was added. The gel was then driedand sealed in plastic for further handling.

Protein Concentration Assays

Protein samples were assayed using the Bio-Rad Protein Assay Reagent.The reagent was diluted 1:4 in water and filtered to remove anyparticulates. 20 mL of protein sample was combined with 980 mL of dilutereagent in a 2 mL spectrophotometer cuvette and allowed to incubate for10-30 minutes. The samples' absorbance was then measured at 595 nm andcompared to that of a sample of known enzyme concentration.

Kinetic Assays

Kinetic assays were performed on three substrates: pNPA, LCN-pNP, andp-nitrobenzyl loracarbef nucleus (LCN-pNB). For pNPA and LCN-pNPsubstrates, final concentrations varying from 0.0625 mM to 16 mM and 0,15, and 30% DMF in 0.1M PIPES (Sigma), pH 7.0 were combined with equalvolumes of enzyme samples. These samples were mixed simultaneously in a96 well plate and monitored with the 96 well plate reader. Theabsorbance values were recorded, linearly regressed, and then used forcalculating kinetic parameters. For the LCN-pNB substrate, finalconcentrations varying from 0.0156 mM to 8.0 mM and 0, 15 and 30% DMF inPIPES, pH 7.0 were combined with enzyme samples in a quartz cuvette andwere measured in a spectrophotometer at 289 nm. All assays were measuredin triplicate.

Additionally, assays on pNB containing substrates were performed byadding a reaction mix containing 1.0 mM substrate in 1 to 20% DMF and0.1M phosphate buffer, pH 7.0 to a small volume of enzyme solution,incubating at room temperature for 20 to 60 minutes, and then stoppingthe reaction with an equal volume of acetonitrile. The samples were theninjected into an HPLC containing a C18 chromatography column andreaction products were separated using a gradient between 95% 1 mMtriethylamine pH 2.5/5% methanol and 100% methanol. The resulting peakswere monitored at 270 nm and recorded on an IBM PC data acquisitionsystem. These peaks were then numerically integrated and used forcomparison between enzyme samples.

The preceding examples demonstrate the usefulness of the presentinvention in preparing, isolating and identifying esterases which haveimproved stability and/or ester hydrolysis activity in organic mediarelative to the natural enzyme.

Having thus described exemplary embodiments of the present invention, itshould be noted by those skilled in the art that the disclosures hereinare exemplary only and that various other alternations, adaptations andmodifications may be made within the scope of the present invention.Accordingly, the present invention is not limited to the specificembodiments as illustrated herein.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 24                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1470 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM:                                                                 (C) INDIVIDUAL ISOLATE: 0- Wtpnb                                              (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: complement (1..1470)                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATGACTCATCAAATAGTAACGACTCAATACGGCAAAGTAAAAGGCACAACGGAAAACGGC60                GTACATAAGTGGAAAGGCATCCCCTATGCCAAGCCGCCTGTCGGACAATGGCGTTTTAAA120               GCACCTGAGCCGCCTGAAGTGTGGGAAGATGTCCTTGATGCCACAGCGTACGGTCCTATT180               TGCCCGCAGCCGTCTGATTTGCTCTCACTGTCGTATACAGAGCTGCCCCGCCAGTCCGAG240               GATTGCTTGTATGTCAATGTATTTGCGCCTGACACTCCAAGTCAAAATCTTCCTGTCATG300               GTGTGGATTCACGGAGGCGCTTTTTATCTTGGAGCGGGCAGTGAGCCATTGTATGACGGA360               TCAAAACTTGCGGCACAGGGAGAAGTCATTGTCGTTACATTGAACTATCGGCTGGGGCCG420               TTTGGCTTTTTGCACTTGTCTTCGTTTGATGAGGCGTATTCCGATAACCTTGGGCTTTTA480               GACCAAGCCGCCGCGCTGAAATGGGTGCGGGAGAATATCTCAGCGTTTGGCGGTGATCCC540               GATAACGTAACAGTATTTGGAGAATCCGCCGGCGGCATGAGCATTGCCGCGCTGCTCGCT600               ATGCCTGCGGCAAAAGGCCTGTTCCAGAAAGCGATCATGGAAAGCGGCGCTTCCCGAACA660               ATGACAAAAGAACAAGCGGCAAGCACTGCGGCTGCCTTTTTACAGGTCCTTGGGATTAAT720               GAGAGCCAGCTGGACAGATTGCATACTGTAGCAGCGGAAGATTTGCTTAAAGCGGCCGAT780               CAGCTTCGGATTGCAGAAAAAGAAAATATCTTTCAGCTGTTCTTCCAGCCCGCCCTTGAT840               CCGAAAACGCTGCCTGAAGAACCAGAAAAATCGATCGCAGAAGGGGCTGCTTCCGGCATT900               CCGCTATTGATTGGAACAACCCGTGATGAAGGATATTTATTTTTCACCCCGGATTCAGAC960               GTTCATTCTCAGGAAACGCTTGATGCAGCACTCGAGTATTTACTAGGGAAGCCGCTGGCA1020              GAGAAAGCTGCCGATTTGTATCCGCGTTCTCTGGAAAGCCAAATTCATATGATGACTGAT1080              TTATTATTTTGGCGCCCTGCCGTCGCCTATGCATCCGCACAGTCTCATTACGCCCCTGTC1140              TGGATGTACCGGTTCGATTGGCACCCGGAGAAGCCGCCGTACAATAAAGCGTTTCACGCA1200              TTAGAGCTTCCTTTTGTCTTTGGAAATCTGGACGGATTGGAACGAATGGCAAAAGCGGAG1260              ATTACGGATGAGGTGAAACAGCTTTCTCACACGATACAATCCGCGTGGATCACGTTCGCT1320              AAAACAGGAAACCCAAGCACCGAAGCTGTGAATTGGCCGGCGTATCATGAAGAAACGAGA1380              GAGACGGTGATTTTAGACTCAGAGATTACGATCGAAAACGATCCCGAATCTGAAAAAAGG1440              CAGAAGCTATTCCCTTCAAAAGGAGAATAA1470                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 489 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: enzyme                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetThrHisGlnIleValThrThrGlnTyrGlyLysValLysGlyThr                              151015                                                                        ThrGluAsnGlyValHisLeuTrpLysGlyIleProTyrAlaLysPro                              202530                                                                        ProValGlyGlnTrpArgPheLysAlaProGluProProGluValTrp                              354045                                                                        GluAspValLeuAspAlaThrAlaTyrGlyProIleCysProGlnPro                              505560                                                                        SerAspLeuLeuSerLeuSerTyrThrGluLeuProArgGlnSerGlu                              65707580                                                                      AspCysLeuTyrValAsnValPheAlaProAspThrProSerGlnAsn                              859095                                                                        LeuProValMetValTrpIleHisGlyGlyAlaPheTyrLeuGlyAla                              100105110                                                                     GlySerGluProLeuTyrAspGlySerLysLeuAlaAlaGlnGlyGlu                              115120125                                                                     ValIleValValThrLeuAsnTyrArgLeuGlyProPheGlyPheLeu                              130135140                                                                     HisLeuSerSerPheAspGluAlaTyrSerAspAsnLeuGlyLeuLeu                              145150155160                                                                  AspGlnAlaAlaAlaLeuLysTrpValArgGluAsnIleSerAlaPhe                              165170175                                                                     GlyGlyAspProAspAsnValThrValPheGlyGluSerAlaGlyGly                              180185190                                                                     MetSerIleAlaAlaLeuLeuAlaMetProAlaAlaLysGlyLeuPhe                              195200205                                                                     GlnLysAlaIleMetGluSerGlyAlaSerArgThrMetThrLysGlu                              210215220                                                                     GlnAlaAlaSerThrAlaAlaAlaPheLeuGlnValLeuGlyIleAsn                              225230235240                                                                  GluSerGlnLeuAspArgLeuHisThrValAlaAlaGluAspLeuLeu                              245250255                                                                     LysAlaAlaAspGlnLeuArgIleAlaGluLysGluAsnIlePheGln                              260265270                                                                     LeuPhePheGlnProAlaLeuAspProLysThrLeuProGluGluPro                              275280285                                                                     GluLysSerIleAlaGluGlyAlaAlaSerGlyIleProLeuLeuIle                              290295300                                                                     GlyThrThrArgAspGluGlyTyrLeuPhePheThrProAspSerAsp                              305310315320                                                                  ValHisSerGlnGluThrLeuAspAlaAlaLeuGluTyrLeuLeuGly                              325330335                                                                     LysProLeuAlaGluLysAlaAlaAspLeuTyrProArgSerLeuGlu                              340345350                                                                     SerGlnIleHisMetMetThrAspLeuLeuPheTrpArgProAlaVal                              355360365                                                                     AlaTyrAlaSerAlaGlnSerHisTyrAlaProValTrpMetTyrArg                              370375380                                                                     PheAspTrpHisProGluLysProProTyrAsnLysAlaPheHisAla                              385390395400                                                                  LeuGluLeuProPheValPheGlyAsnLeuAspGlyLeuGluArgMet                              405410415                                                                     AlaLysAlaGluIleThrAspGluValLysGlnLeuSerHisThrIle                              420425430                                                                     GlnSerAlaTrpIleThrPheAlaLysThrGlyAsnProSerThrGlu                              435440445                                                                     AlaValAsnTrpProAlaTyrHisGluGluThrArgGluThrValIle                              450455460                                                                     LeuAspSerGluIleThrIleGluAsnAspProGluSerGluLysArg                              465470475480                                                                  GlnLysLeuPheProSerLysGlyGlu                                                   485                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1470 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM:                                                                 (C) INDIVIDUAL ISOLATE: 1-1h9                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: complement (1..1470)                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ATGACTCATCAAATAGTAACGACTCAATACGGCAAAGTAAAAGGCACAACGGAAAACGGC60                GTACATAAGTGGAAAGGCATCCCTTATGCCAAGCCGCCTGTCGGACAATGGCGTTTTAAA120               GCACCTGAGCCGCCTGAAGTGTGGGAAGATGTCCTTGATGCCACAGCGTACGGTCCTATT180               TGCCCGCAGCCGTCTGATTTGCTCTCACTGTCGTATACAGAGCTGCCCCGCCAGTCCGAG240               GATTGCTTGTATGTCAATGTATTTGCGCCTGACACTCCAAGTCAAAATCTTCCTGTCATG300               GTGTGGATTCACGGAGGCGCTTTTTATCTTGGAGCGGGCAGTGAGCCATTGTATGACGGA360               TCAAAACTTGCGGCACAGGGAGAAGTCATTGTCGTTACATTGAACTATCGGCTGGGGCCG420               TTTGGCTTTTTGCACTTGTCTTCGTTTGATGAGGCGTATTCCGATAACCTTGGGCTTTTA480               GACCAAGCCGCCGCGCTGAAATGGGTGCGGGAGAATATCTCAGCGTTTGGCGGTGATCCC540               GATAACGTAACAGTATTTGGAGAATCCGCCGGCGGCATGAGCATTGCCGCGCTGCTCGCT600               ATGCCTGCGGCAAAAGGCCTGTTCCAGAAAGCGATCATGGAAAGCGGCGCTTCCCGAACA660               ATGACAAAAGAACAAGCGGCAAGCACTGCGGCTGCCTTTTTACAGGTCCTTGGGATTAAT720               GAGAGCCAGCTGGACAGATTGCATACTGTAGCAGCGGAAGATTTGCTTAAAGCGGCCGAT780               CAGCTTCGGATTGCAGAAAAAGAAAATATCTTTCAGCTGTTCTTCCAGCCCGCCCTTGAT840               CCGAAAACGCTGCCTGAAGAACCAGAAAAATCGATCGCAGAAGGGGCTGCTTCCGGCATT900               CCGCTATTGATTGGAACAACCCGTGATGAAGGATATTTATTTTTCACCCCGGATTCAGAC960               GTTCGTTCTCAGGAAACGCTTGATGCAGCACTCGAGTATTTACTAGGGAAGCCGCTGGCA1020              GAGAAAGCTGCCGATTTGTATCCGCGTTCTCTGGAAAGCCAAATTCATATGATGACTGAT1080              TTATTATTTTGGCGCCCTGCCGTCGCCTTTGCATCCGCACAGTCTCATTACGCCCCTGTC1140              TGGATGTACCGGTTCGATTGGCACCCGGAGAAGCCGCCGTACAATAAAGCGTTTCACGCA1200              TTAGAGCTTCCTTTTGTCTTTGGAAATCTGGACGGATTGGAACGAATGGCAAAAGCGGAG1260              ATTACGGATGAGGTGAAACAGCTTTCTCACACGATACAATCCGCGTGGATCACGTTCGCT1320              AAAACAGGAAACCCAAGCACCGAAGCTGTGAATTGGCCGGCGTATCATGAAGAAACGAGA1380              GAGACGGTGATTTTAGACTCAGAGATTACGATCGAAAACGATCCCGAATCTGAAAAAAGG1440              CAGAAGCTATTCCCTTCAAAAGGAGAATAA1470                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 489 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: enzyme                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetThrHisGlnIleValThrThrGlnTyrGlyLysValLysGlyThr                              151015                                                                        ThrGluAsnGlyValHisLeuTrpLysGlyIleProTyrAlaLysPro                              202530                                                                        ProValGlyGlnTrpArgPheLysAlaProGluProProGluValTrp                              354045                                                                        GluAspValLeuAspAlaThrAlaTyrGlyProIleCysProGlnPro                              505560                                                                        SerAspLeuLeuSerLeuSerTyrThrGluLeuProArgGlnSerGlu                              65707580                                                                      AspCysLeuTyrValAsnValPheAlaProAspThrProSerGlnAsn                              859095                                                                        LeuProValMetValTrpIleHisGlyGlyAlaPheTyrLeuGlyAla                              100105110                                                                     GlySerGluProLeuTyrAspGlySerLysLeuAlaAlaGlnGlyGlu                              115120125                                                                     ValIleValValThrLeuAsnTyrArgLeuGlyProPheGlyPheLeu                              130135140                                                                     HisLeuSerSerPheAspGluAlaTyrSerAspAsnLeuGlyLeuLeu                              145150155160                                                                  AspGlnAlaAlaAlaLeuLysTrpValArgGluAsnIleSerAlaPhe                              165170175                                                                     GlyGlyAspProAspAsnValThrValPheGlyGluSerAlaGlyGly                              180185190                                                                     MetSerIleAlaAlaLeuLeuAlaMetProAlaAlaLysGlyLeuPhe                              195200205                                                                     GlnLysAlaIleMetGluSerGlyAlaSerArgThrMetThrLysGlu                              210215220                                                                     GlnAlaAlaSerThrAlaAlaAlaPheLeuGlnValLeuGlyIleAsn                              225230235240                                                                  GluSerGlnLeuAspArgLeuHisThrValAlaAlaGluAspLeuLeu                              245250255                                                                     LysAlaAlaAspGlnLeuArgIleAlaGluLysGluAsnIlePheGln                              260265270                                                                     LeuPhePheGlnProAlaLeuAspProLysThrLeuProGluGluPro                              275280285                                                                     GluLysSerIleAlaGluGlyAlaAlaSerGlyIleProLeuLeuIle                              290295300                                                                     GlyThrThrArgAspGluGlyTyrLeuPhePheThrProAspSerAsp                              305310315320                                                                  ValArgSerGlnGluThrLeuAspAlaAlaLeuGluTyrLeuLeuGly                              325330335                                                                     LysProLeuAlaGluLysAlaAlaAspLeuTyrProArgSerLeuGlu                              340345350                                                                     SerGlnIleHisMetMetThrAspLeuLeuPheTrpArgProAlaVal                              355360365                                                                     AlaPheAlaSerAlaGlnSerHisTyrAlaProValTrpMetTyrArg                              370375380                                                                     PheAspTrpHisProGluLysProProTyrAsnLysAlaPheHisAla                              385390395400                                                                  LeuGluLeuProPheValPheGlyAsnLeuAspGlyLeuGluArgMet                              405410415                                                                     AlaLysAlaGluIleThrAspGluValLysGlnLeuSerHisThrIle                              420425430                                                                     GlnSerAlaTrpIleThrPheAlaLysThrGlyAsnProSerThrGlu                              435440445                                                                     AlaValAsnTrpProAlaTyrHisGluGluThrArgGluThrValIle                              450455460                                                                     LeuAspSerGluIleThrIleGluAsnAspProGluSerGluLysArg                              465470475480                                                                  GlnLysLeuPheProSerLysGlyGlu                                                   485                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1470 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM:                                                                 (C) INDIVIDUAL ISOLATE: 2- 19E10                                              (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: complement (1..1470)                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ATGACTCATCAAATAGTAACGACTCAATACGGCAAAGTAAAAGGCACAACGGAAAACGGC60                GTACATAAGTGGAAAGGCATCCCTTATGCCAAGCCGCCTGTCGGACAATGGCGTTTTAAA120               GCACCTGAGCCGCCTGAAGTGTGGGAAGATGTCCTTGATGCCACAGCGTACGGTCCTATT180               TGCCCGCAGCCGTCTGATTTGCTCTCACTGTCGTATACAGAGCTGCCCCGCCAGTCCGAG240               GATTGCTTGTATGTCAATGTATTTGCGCCTGACACTCCAAGTCAAAATCTTCCTGTCATG300               GTGTGGATTCACGGAGGCGCTTTTTATCTAGGAGCGGGCAGTGAGCCATTGTATGACGGA360               TCAAAACTTGCGGCACAGGGAGAAGTCATTGTCGTTACATTGAACTATCGGCTGGGGCCG420               TTTGGCTTTTTGCACTTGTCTTCGTTTGATGAGGCGTATTCCGATAACCTTGGGCTTTTA480               GACCAAGCCGCCGCGCTGAAATGGGTGCGGGAGAATATCTCAGCGTTTGGCGGTGATCCC540               GATAACGTAACAGTATTTGGAGAATCCGCCGGCGGCATGAGCATTGCCGCGCTGCTCGCT600               ATGCCTGCGGCAAAAGGCCTGTTCCAGAAAGCGATCATGGAAAGCGGCGCTTCCCGAACA660               ATGACAAAAGAACAAGCGGCAAGCACTGCGGCTGCCTTTTTACAGGTCCTTGGGATTAAT720               GAGAGCCAGCTGGACAGATTGCATACTGTAGCAGCGGAAGATTTGCTTAAAGCGGCCGAT780               CAGCTTCGGATTGCAGAAAAAGAAAATATCTTTCAGCTGTTCTTCCAGCCCGCCCTTGAT840               CCGAAAACGCTGCCTGAAGAACCAGAAAAATCGATCGCAGAAGGGGCTGCTTCCGGCATT900               CCGCTATTGATTGGAACAACCCGTGATGAAGGATATTTATTTTTCACCCCGGATTCAGAC960               GTTCGTTCTCAGGAAACGCTTGATGCAGCACTCGAGTATTTACTAGGGAAGCCGCTGGCA1020              GAGAAAGCTGCCGATTTGTATCCGCGTTCTCTGGAAAGCCAAATTCATATGGTGACTGAT1080              TTATTATTTTGGCGCCCTGCCGTCGCCTTTGCATCCGCACAGTCTCATTACGCCCCTGTC1140              TGGATGTACCGGTTCGATTGGCACCCGGAGAAGCCGCCGTACAATAAAGCGTTTCACGCA1200              TTAGAGCTTCCTTTTGTCTTTGGAAATCTGGACGGGTTGGAACGAATGGCAAAAGCGGAG1260              ATTACGGATGAGGTGAAACAGCTTTCTCACACGATACAATCCGCGTGGATCACGTTCGCT1320              AAAACAGGAAACCCAAGCACCGAAGCTGTGAATTGGCCGGCGTATCATGAAGAAACGAGA1380              GAGACGGTGATTTTAGACTCAGAGATTACGATCGAAAACGATCCCGAATCTGAAAAAAGG1440              CAGAAGCTATTCCCTTCAAAAGGAGAATAA1470                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 489 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: enzyme                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetThrHisGlnIleValThrThrGlnTyrGlyLysValLysGlyThr                              151015                                                                        ThrGluAsnGlyValHisLeuTrpLysGlyIleProTyrAlaLysPro                              202530                                                                        ProValGlyGlnTrpArgPheLysAlaProGluProProGluValTrp                              354045                                                                        GluAspValLeuAspAlaThrAlaTyrGlyProIleCysProGlnPro                              505560                                                                        SerAspLeuLeuSerLeuSerTyrThrGluLeuProArgGlnSerGlu                              65707580                                                                      AspCysLeuTyrValAsnValPheAlaProAspThrProSerGlnAsn                              859095                                                                        LeuProValMetValTrpIleHisGlyGlyAlaPheTyrLeuGlyAla                              100105110                                                                     GlySerGluProLeuTyrAspGlySerLysLeuAlaAlaGlnGlyGlu                              115120125                                                                     ValIleValValThrLeuAsnTyrArgLeuGlyProPheGlyPheLeu                              130135140                                                                     HisLeuSerSerPheAspGluAlaTyrSerAspAsnLeuGlyLeuLeu                              145150155160                                                                  AspGlnAlaAlaAlaLeuLysTrpValArgGluAsnIleSerAlaPhe                              165170175                                                                     GlyGlyAspProAspAsnValThrValPheGlyGluSerAlaGlyGly                              180185190                                                                     MetSerIleAlaAlaLeuLeuAlaMetProAlaAlaLysGlyLeuPhe                              195200205                                                                     GlnLysAlaIleMetGluSerGlyAlaSerArgThrMetThrLysGlu                              210215220                                                                     GlnAlaAlaSerThrAlaAlaAlaPheLeuGlnValLeuGlyIleAsn                              225230235240                                                                  GluSerGlnLeuAspArgLeuHisThrValAlaAlaGluAspLeuLeu                              245250255                                                                     LysAlaAlaAspGlnLeuArgIleAlaGluLysGluAsnIlePheGln                              260265270                                                                     LeuPhePheGlnProAlaLeuAspProLysThrLeuProGluGluPro                              275280285                                                                     GluLysSerIleAlaGluGlyAlaAlaSerGlyIleProLeuLeuIle                              290295300                                                                     GlyThrThrArgAspGluGlyTyrLeuPhePheThrProAspSerAsp                              305310315320                                                                  ValArgSerGlnGluThrLeuAspAlaAlaLeuGluTyrLeuLeuGly                              325330335                                                                     LysProLeuAlaGluLysAlaAlaAspLeuTyrProArgSerLeuGlu                              340345350                                                                     SerGlnIleHisMetValThrAspLeuLeuPheTrpArgProAlaVal                              355360365                                                                     AlaPheAlaSerAlaGlnSerHisTyrAlaProValTrpMetTyrArg                              370375380                                                                     PheAspTrpHisProGluLysProProTyrAsnLysAlaPheHisAla                              385390395400                                                                  LeuGluLeuProPheValPheGlyAsnLeuAspGlyLeuGluArgMet                              405410415                                                                     AlaLysAlaGluIleThrAspGluValLysGlnLeuSerHisThrIle                              420425430                                                                     GlnSerAlaTrpIleThrPheAlaLysThrGlyAsnProSerThrGlu                              435440445                                                                     AlaValAsnTrpProAlaTyrHisGluGluThrArgGluThrValIle                              450455460                                                                     LeuAspSerGluIleThrIleGluAsnAspProGluSerGluLysArg                              465470475480                                                                  GlnLysLeuPheProSerLysGlyGlu                                                   485                                                                           (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1470 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM:                                                                 (C) INDIVIDUAL ISOLATE: 3-10c4                                                (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: complement (1..1470)                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ATGACTCATCAAATAGTAACGACTCAATACGGCAAAGTAAAAGGCACAACGGAAAACGGC60                GTACATAAGTGGAAAGGCATCCCTTATGCCAAGCCGCCTGTCGGACAATGGCGTTTTAAA120               GCACCTGAGCCGCCTGAAGTGTGGGAAGATGTCCTTGATGCCACAGCGTACGGTCCTATT180               TGCCCGCAGCCGTCTGATTTGCTCTCACTGTCGTATACAGAGCTGCCCCGCCAGTCCGAG240               GATTGCTTGTATGTCAATGTATTTGCGCCTGACACTCCAAGTCAAAACCTTCCTGTCATG300               GTGTGGATTCACGGAGGCGCTTTTTATCTAGGAGCGGGCAGTGAGCCATTGTATGACGGA360               TCAAAACTTGCGGCACAGGGAGAAGTCATTGTCGTTACATTGAACTATCGGCTGGGGCCG420               TTTGGCTTTATGCACTTGTCTTCGTTTGATGAGGCGTATTCCGATAACCTTGGGCTTTTA480               GACCAAGCCGCCGCGCTGAAATGGGTGCGGGAGAATATCTCAGCGTTTGGCGGTGATCCC540               GATAACGTAACAGTATTTGGAGAATCCGCCGGCGGCATGAGCATTGCCGCGCTGCTCGCT600               ATGCCTGCGGCAAAAGGCCTGTTCCAGAAAGCGATCATGGAAAGCGGCGCTTCCCGAACA660               ATGACAAAAGAACAAGCGGCAAGCACTGCGGCTGCCTTTTTACAGGTCCTTGGGATTAAT720               GAGAGCCAGCTGGACAGATTGCATACTGTAGCAGCGGAAGATTTGCTTAAAGCGGCCGAT780               CAGCTTCGGATTGCAGAAAAAGAAAATATCTTTCAGCTGTTCTTCCAGCCCGCCCTTGAT840               CCGAAAACGCTGCCTGAAGAACCAGAAAAATCGATCGCAGAAGGGGCTGCTTCCGGCATT900               CCGCTATTGATTGGAACAACCCGTGATGAAGGATATTTATTTTTCACCCCGGATTCAGAC960               GTTCGTTCTCAGGAAACGCTTGATGCAGCACTCGAGTATTTACTAGGGAAGCCGCTGGCA1020              GAGAAAGCTGCCGATTTGTATCCGCGTTCTCTGGAAAGCCAAATTCATATGGTGACTGAT1080              TTATTATTTTGGCGCCCTGCCGTCGCCTTTGCATCCGCACAGTCTCATTACGCCCCTGTC1140              TGGATGTACCGGTTCGATTGGCACCCGGAGAAGCCGCCGTACAATAAAGCGTTTCACGCA1200              TTAGAGCTTCCTTTTGTCTTTGGAAATCTGGACGGGTTGGAACGAATGGCAAAAGCGGAG1260              ATTACGGATGAGGTGAAACAGCTTTCTCACACGATACAATCCGCGTGGATCACGTTCGCT1320              AAAACAGGAAACCCAAGCACCGAAGCTGTGAATTGGCCGGCGTATCATGAAGAAACGAGA1380              GAGACGGTGATTTTAGACTCAGAGATTACGATCGAAAACGATCCCGAATCTGAAAAAAGG1440              CAGAAGCTATTCCCTTCAAAAGGAGAATAA1470                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 489 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: enzyme                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       MetThrHisGlnIleValThrThrGlnTyrGlyLysValLysGlyThr                              151015                                                                        ThrGluAsnGlyValHisLeuTrpLysGlyIleProTyrAlaLysPro                              202530                                                                        ProValGlyGlnTrpArgPheLysAlaProGluProProGluValTrp                              354045                                                                        GluAspValLeuAspAlaThrAlaTyrGlyProIleCysProGlnPro                              505560                                                                        SerAspLeuLeuSerLeuSerTyrThrGluLeuProArgGlnSerGlu                              65707580                                                                      AspCysLeuTyrValAsnValPheAlaProAspThrProSerGlnAsn                              859095                                                                        LeuProValMetValTrpIleHisGlyGlyAlaPheTyrLeuGlyAla                              100105110                                                                     GlySerGluProLeuTyrAspGlySerLysLeuAlaAlaGlnGlyGlu                              115120125                                                                     ValIleValValThrLeuAsnTyrArgLeuGlyProPheGlyPheMet                              130135140                                                                     HisLeuSerSerPheAspGluAlaTyrSerAspAsnLeuGlyLeuLeu                              145150155160                                                                  AspGlnAlaAlaAlaLeuLysTrpValArgGluAsnIleSerAlaPhe                              165170175                                                                     GlyGlyAspProAspAsnValThrValPheGlyGluSerAlaGlyGly                              180185190                                                                     MetSerIleAlaAlaLeuLeuAlaMetProAlaAlaLysGlyLeuPhe                              195200205                                                                     GlnLysAlaIleMetGluSerGlyAlaSerArgThrMetThrLysGlu                              210215220                                                                     GlnAlaAlaSerThrAlaAlaAlaPheLeuGlnValLeuGlyIleAsn                              225230235240                                                                  GluSerGlnLeuAspArgLeuHisThrValAlaAlaGluAspLeuLeu                              245250255                                                                     LysAlaAlaAspGlnLeuArgIleAlaGluLysGluAsnIlePheGln                              260265270                                                                     LeuPhePheGlnProAlaLeuAspProLysThrLeuProGluGluPro                              275280285                                                                     GluLysSerIleAlaGluGlyAlaAlaSerGlyIleProLeuLeuIle                              290295300                                                                     GlyThrThrArgAspGluGlyTyrLeuPhePheThrProAspSerAsp                              305310315320                                                                  ValArgSerGlnGluThrLeuAspAlaAlaLeuGluTyrLeuLeuGly                              325330335                                                                     LysProLeuAlaGluLysAlaAlaAspLeuTyrProArgSerLeuGlu                              340345350                                                                     SerGlnIleHisMetValThrAspLeuLeuPheTrpArgProAlaVal                              355360365                                                                     AlaPheAlaSerAlaGlnSerHisTyrAlaProValTrpMetTyrArg                              370375380                                                                     PheAspTrpHisProGluLysProProTyrAsnLysAlaPheHisAla                              385390395400                                                                  LeuGluLeuProPheValPheGlyAsnLeuAspGlyLeuGluArgMet                              405410415                                                                     AlaLysAlaGluIleThrAspGluValLysGlnLeuSerHisThrIle                              420425430                                                                     GlnSerAlaTrpIleThrPheAlaLysThrGlyAsnProSerThrGlu                              435440445                                                                     AlaValAsnTrpProAlaTyrHisGluGluThrArgGluThrValIle                              450455460                                                                     LeuAspSerGluIleThrIleGluAsnAspProGluSerGluLysArg                              465470475480                                                                  GlnLysLeuPheProSerLysGlyGlu                                                   485                                                                           (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1470 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM:                                                                 (C) INDIVIDUAL ISOLATE: 4-38b9                                                (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: complement (1..1470)                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       ATGACTCATCAAATAGTAACGACTCAATACGGCAAAGTAAAAGGCACAACGGAAAACGGC60                GTACATAAGTGGAAAGGCATCCCTTATGCCAAGCCGCCTGTCGGACAATGGCGTTTTAAA120               GCACCTGAGCCGCCTGAAGTGTGGGAAGATGTCCTTGATGCCACAGCGTACGGTCCTGTT180               TGCCCGCAGCCGTCTGATTTGCTCTCACTGTCGTATACAGAGCTGCCCCGCCAGTCCGAG240               GATTGCTTGTATGTCAATGTATTTGCGCCTGACACTCCAAGTCAAAACCTTCCTGTCATG300               GTGTGGATTCACGGAGGCGCTTTTTATCTAGGAGCGGGCAGTGAGCCATTGTATGACGGA360               TCAAAACTTGCGGCACAGGGAGAAGTCATTGTCGTTACATTGAACTATCGGCTGGGGCCG420               TTTGGCTTTATGCACTTGTCTTCGTTTGATGAGGCGTATTCCGATAACCTTGGGCTTTTA480               GACCAAGCCGCCGCGCTGAAATGGGTGCGGGAGAATATCTCAGCGTTTGGCGGTGATCCC540               GATAACGTAACAGTATTTGGAGAATCCGCCGGCGGCATGAGCATTGCCGCGCTGCTCGCT600               ATGCCTGCGGCAAAAGGCCTGTTCCAGAAAGCGATCATGGAAAGCGGCGCTTCCCGAACA660               ATGACAAAAGAACAAGCGGCAAGCACTGCGGCTGCCTTTTTACAGGTCCTTGGGATTAAT720               GAGAGCCAGCTGGACAGATTGCATACTGTAGCAGCGGAAGATTTGCTTAAAGCGGCCGAT780               CAGCTTCGGATTGCAGAAAAAGAAAATATCTTTCAGCTGTTCTTCCAGCCCGCCCTTGAT840               CCGAAAACGCTGCCTGAAGAACCAGAAAAATCGATCGCAGAAGGGGCTGCTTCCGGCATT900               CCGCTATTGATTGGAACAACCCGTGATGAAGGATATTTATTTTTCACCCCGGATTCAGAC960               GTTCGTTCTCAGGAAACGCTTGATGCAGCACTCGAGTATTTACTAGGGAAGCCGCTGGCA1020              GAGAAAGCTGCCGATTTGTATCCGCGTTCTCTGGAAAGCCAAATTCATATGGTGACTGAT1080              TTATTATTTTGGCGCCCTGCCGTCGCCTTTGCATCCGCACAGTCTCATTACGCCCCTGTC1140              TGGATGTACCGGTTCGATTGGCACCCGGAGAAGCCGCCGTACAATAAAGCGTTTCACGCA1200              TTAGAGCTTCCTTTTGTCTTTGGAAATCTGGACGGGTTGGAACGAATGGCAAAAGCGGAG1260              ATTACGGATGAGGTGAAACAGCTTTCTCACACGATACAATCCGCGTGGATCACGTTCGCT1320              AAAACAGGAAACCCAAGCACCGAAGCTGTGAATTGGCCGGCGTATCATGAAGAAACGAGA1380              GAGACGGTGATTTTAGACTCAGAGATTACGATCGAAAACGATCCCGAATCTGAAAAAAGG1440              CAGAAGCTATTCCCTTCAAAAGGAGAATAA1470                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 489 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: enzyme                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      MetThrHisGlnIleValThrThrGlnTyrGlyLysValLysGlyThr                              151015                                                                        ThrGluAsnGlyValHisLeuTrpLysGlyIleProTyrAlaLysPro                              202530                                                                        ProValGlyGlnTrpArgPheLysAlaProGluProProGluValTrp                              354045                                                                        GluAspValLeuAspAlaThrAlaTyrGlyProValCysProGlnPro                              505560                                                                        SerAspLeuLeuSerLeuSerTyrThrGluLeuProArgGlnSerGlu                              65707580                                                                      AspCysLeuTyrValAsnValPheAlaProAspThrProSerGlnAsn                              859095                                                                        LeuProValMetValTrpIleHisGlyGlyAlaPheTyrLeuGlyAla                              100105110                                                                     GlySerGluProLeuTyrAspGlySerLysLeuAlaAlaGlnGlyGlu                              115120125                                                                     ValIleValValThrLeuAsnTyrArgLeuGlyProPheGlyPheMet                              130135140                                                                     HisLeuSerSerPheAspGluAlaTyrSerAspAsnLeuGlyLeuLeu                              145150155160                                                                  AspGlnAlaAlaAlaLeuLysTrpValArgGluAsnIleSerAlaPhe                              165170175                                                                     GlyGlyAspProAspAsnValThrValPheGlyGluSerAlaGlyGly                              180185190                                                                     MetSerIleAlaAlaLeuLeuAlaMetProAlaAlaLysGlyLeuPhe                              195200205                                                                     GlnLysAlaIleMetGluSerGlyAlaSerArgThrMetThrLysGlu                              210215220                                                                     GlnAlaAlaSerThrAlaAlaAlaPheLeuGlnValLeuGlyIleAsn                              225230235240                                                                  GluSerGlnLeuAspArgLeuHisThrValAlaAlaGluAspLeuLeu                              245250255                                                                     LysAlaAlaAspGlnLeuArgIleAlaGluLysGluAsnIlePheGln                              260265270                                                                     LeuPhePheGlnProAlaLeuAspProLysThrLeuProGluGluPro                              275280285                                                                     GluLysSerIleAlaGluGlyAlaAlaSerGlyIleProLeuLeuIle                              290295300                                                                     GlyThrThrArgAspGluGlyTyrLeuPhePheThrProAspSerAsp                              305310315320                                                                  ValArgSerGlnGluThrLeuAspAlaAlaLeuGluTyrLeuLeuGly                              325330335                                                                     LysProLeuAlaGluLysAlaAlaAspLeuTyrProArgSerLeuGlu                              340345350                                                                     SerGlnIleHisMetValThrAspLeuLeuPheTrpArgProAlaVal                              355360365                                                                     AlaPheAlaSerAlaGlnSerHisTyrAlaProValTrpMetTyrArg                              370375380                                                                     PheAspTrpHisProGluLysProProTyrAsnLysAlaPheHisAla                              385390395400                                                                  LeuGluLeuProPheValPheGlyAsnLeuAspGlyLeuGluArgMet                              405410415                                                                     AlaLysAlaGluIleThrAspGluValLysGlnLeuSerHisThrIle                              420425430                                                                     GlnSerAlaTrpIleThrPheAlaLysThrGlyAsnProSerThrGlu                              435440445                                                                     AlaValAsnTrpProAlaTyrHisGluGluThrArgGluThrValIle                              450455460                                                                     LeuAspSerGluIleThrIleGluAsnAspProGluSerGluLysArg                              465470475480                                                                  GlnLysLeuPheProSerLysGlyGlu                                                   485                                                                           (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1470 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM:                                                                 (C) INDIVIDUAL ISOLATE: 4-43e7                                                (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: complement (1..1470)                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      ATGACTCATCAAATAGTAACGACTCAATACGGCAAAGTAAAAGGCACAACGGAAAACGGC60                GTACATAAGTGGAAAGGCATCCCTTATGCCAAGCCGCCTGTCGGACAATGGCGTTTTAAA120               GCACCTGAGCCGCCTGAAGTGTGGGAAGATGTCCTTGATGCCACAGCGTACGGTCCTATT180               TGCCCGCAGCCGTCTGATTTGCTCTCACTGTCGTATACAGAGCTGCCCCGCCAGTCCGAG240               GATTGCTTGTATGTCAATGTATTTGCGCCTGACACTCCAGGTCAAAACCTTCCTGTCATG300               GTGTGGATTCACGGAGGCGCTTTTTATCTAGGAGCGGGCAGTGAGCCATTGTATGACGGA360               TCAAAACTTGCGGCACAGGGAGAAGTCATTGTCGTTACATTGAACTATCGGCTGGGGCCG420               TTTGGCTTTATGCACTTGTCTTCGTTTGATGAGGCGTATTCCGATAACCTTGGGCTTTTA480               GACCAAGCCGCCGCGCTGAAATGGGTGCGGGAGAATATCTCAGCGTTTGGCGGTGATCCC540               GATAACGTAACAGTATTTGGAGAATCCGCCGGCGGCATGAGCATTGCCGCGCTGCTCGCT600               ATGCCTGCGGCAAAAGGCCTGTTCCAGAAAGCGATCATGGAAAGCGGCGCTTCCCGAACA660               ATGACAAAAGAACAAGCGGCAAGCACTGCGGCTGCCTTTTTACAGGTCCTTGGGATTAAT720               GAGAGCCAGCTGGACAGATTGCATACTGTAGCAGCGGAAGATTTGCTTAAAGCGGCCGAT780               CAGCTTCGGATTGCAGAAAAAGAAAATATCTTTCAGCTGTTCTTCCAGCCCGCCCTTGAT840               CCGAAAACGCTGCCTGAAGAACCAGAAAAATCGATCGCAGAAGGGGCTGCTTCCGGCATT900               CCGCTATTGATTGGAACAACCCGTGATGAAGGATATTTATTTTTCACCCCGGATTCAGAC960               GTTCGTTCTCAGGAAACGCTTGATGCAGCACTCGAGTATTTACTAGGGAAGCCGCTGGCA1020              GAGAAAGCTGCCGATTTGTATCCGCGTTCTCTGGAAAGCCAAATTCATATGGTGACTGAT1080              TTATTATTTTGGCGCCCTGCCGTCGCCTTTGCATCCGCACAGTCTCATTACGCCCCTGTC1140              TGGATGTACCGGTTCGATTGGCACCCGGAGAAGCCGCCGTACAATAAAGCGTTTCACGCA1200              TTAGAGCTTCCTTTTGTCTTTGGAAATCTGGACGGGTTGGAACGAATGGCAAAAGCGGAG1260              ATTACGGATGAGGTGAAACAGCTTTCTCACACGATACAATCCGCGTGGATCACGTTCGCT1320              AAAACAGGAAACCCAAGCACCGAAGCTGTGAATTGGCCGGCGTATCATGAAGAAACGAGA1380              GAGACGGTGATTTTAGACTCAGAGATTACGATCGAAAACGATCCCGAATCTGAAAAAAGG1440              CAGAAGCTATTCCCTTCAAAAGGAGAATAA1470                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 489 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: enzyme                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      MetThrHisGlnIleValThrThrGlnTyrGlyLysValLysGlyThr                              151015                                                                        ThrGluAsnGlyValHisLeuTrpLysGlyIleProTyrAlaLysPro                              202530                                                                        ProValGlyGlnTrpArgPheLysAlaProGluProProGluValTrp                              354045                                                                        GluAspValLeuAspAlaThrAlaTyrGlyProIleCysProGlnPro                              505560                                                                        SerAspLeuLeuSerLeuSerTyrThrGluLeuProArgGlnSerGlu                              65707580                                                                      AspCysLeuTyrValAsnValPheAlaProAspThrProGlyGlnAsn                              859095                                                                        LeuProValMetValTrpIleHisGlyGlyAlaPheTyrLeuGlyAla                              100105110                                                                     GlySerGluProLeuTyrAspGlySerLysLeuAlaAlaGlnGlyGlu                              115120125                                                                     ValIleValValThrLeuAsnTyrArgLeuGlyProPheGlyPheMet                              130135140                                                                     HisLeuSerSerPheAspGluAlaTyrSerAspAsnLeuGlyLeuLeu                              145150155160                                                                  AspGlnAlaAlaAlaLeuLysTrpValArgGluAsnIleSerAlaPhe                              165170175                                                                     GlyGlyAspProAspAsnValThrValPheGlyGluSerAlaGlyGly                              180185190                                                                     MetSerIleAlaAlaLeuLeuAlaMetProAlaAlaLysGlyLeuPhe                              195200205                                                                     GlnLysAlaIleMetGluSerGlyAlaSerArgThrMetThrLysGlu                              210215220                                                                     GlnAlaAlaSerThrAlaAlaAlaPheLeuGlnValLeuGlyIleAsn                              225230235240                                                                  GluSerGlnLeuAspArgLeuHisThrValAlaAlaGluAspLeuLeu                              245250255                                                                     LysAlaAlaAspGlnLeuArgIleAlaGluLysGluAsnIlePheGln                              260265270                                                                     LeuPhePheGlnProAlaLeuAspProLysThrLeuProGluGluPro                              275280285                                                                     GluLysSerIleAlaGluGlyAlaAlaSerGlyIleProLeuLeuIle                              290295300                                                                     GlyThrThrArgAspGluGlyTyrLeuPhePheThrProAspSerAsp                              305310315320                                                                  ValArgSerGlnGluThrLeuAspAlaAlaLeuGluTyrValLeuGly                              325330335                                                                     LysProLeuAlaGluLysAlaAlaAspLeuTyrProArgSerLeuGlu                              340345350                                                                     SerGlnIleHisMetValThrAspLeuLeuPheTrpArgProAlaVal                              355360365                                                                     AlaPheAlaSerAlaGlnSerHisTyrAlaProValTrpMetTyrArg                              370375380                                                                     PheAspTrpHisProGluLysProProTyrAsnLysAlaPheHisAla                              385390395400                                                                  LeuGluLeuProPheValPheGlyAsnLeuAspGlyLeuGluArgMet                              405410415                                                                     AlaLysAlaGluIleThrAspGluValLysGlnLeuSerHisThrIle                              420425430                                                                     GlnSerAlaTrpIleThrPheAlaLysThrGlyAsnProSerThrGlu                              435440445                                                                     AlaValAsnTrpProAlaTyrHisGluGluThrArgGluThrValIle                              450455460                                                                     LeuAspSerGluIleThrIleGluAsnAspProGluSerGluLysArg                              465470475480                                                                  GlnLysLeuPheProSerLysGlyGlu                                                   485                                                                           (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1470 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM:                                                                 (C) INDIVIDUAL ISOLATE: 4-54b9                                                (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: complement (1..1470)                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      ATGACTCATCAAATAGTAACGACTCAATACGGCAAAGTAAAAGGCACAACGGAAAACGGC60                GTACATAAGTGGAAAGGCATCCCTTATGCCAAGCCGCCCGTCGGACAATGGCGTTTTAAA120               GCACCTGAGCCGCCTGAAGTGTGGGAAGATGTCCTTGATGCCACAGCGTACGGTCCTATT180               TGCCCGCAGCCGTCTGATTTGCTCTCACTGTCGTATACAGAGCTGCCCCGCCAGTCCGAG240               GATTGCTTGTATGTCAATGTATTTGCGCCTGACACTCCAAGTCAAAACCTTCCTGTCATG300               GTGTGGATTCACGGAGGCGCTTTTTATCTAGGAGCGGGCAGTGAGCCATTGTATGACGGA360               TCAAAACTTGCGGCACAGGGAGAAGTCATTGTCGTTACATTGAACTATCGGCTGGGGCCG420               TTTGGCTTTATGCACTTGTCTTCGTTTGATGAGGCGTATTCCGATAACCTTGGGCTTTTA480               GACCAAGCCGCCGCGCTGAAATGGGTGCGGGAGAATATCTCAGCGTTTGGCGGTGATCCC540               GATAACGTAACAGTATTTGGAGAATCCGCCGGCGGCATGAGCATTGCCGCGCTGCTCGCT600               ATGCCTGCGGCAAAAGGCCTGTTCCAGAAAGCGATCATGGAAAGCGGCGCTTCCCGAACA660               ATGACAAAAGAACAAGCGGCAAGCACTGCGGCTGCCTTTTTACAGGTCCTTGGGATTAAT720               GAGAGCCAGCTGGACAGATTGCATACTGTAGCAGCGGAAGATTTGCTTAAAGCGGCCGAT780               CAGCTTCGGATTGCAGAAAAAGAAAATATCTTTCAGCTGTTCTTCCAGCCCGCCCTTGAT840               CCGAAAACGCTGCCTGAAGAACCAGAAAAATCGATCGCAGAAGGGGCTGCTTCCGGCATT900               CCGCTATTGATTGGAACAACCCGTGATGAAGGATATTTATTTTTCACCCCGGATTCAGAC960               GTTCGTTCTCAGGAAACGCTTGATGCAGCACTCGAGTATTTACTAGGGAAGCCGCTGGCA1020              GAGAAAGCTGCCGATTTGTATCCGCGTTCTCTGGAAAGCCAAATTCATATGGTGACTGAT1080              TTATTATTTTGGCGCCCTGCCGTCGCCTTTGCATCCGCACAGTCTCATTACGCCCCTGTC1140              TGGATGTACCGGTTCGATTGGCACCCGGAGAAGCCGCCGTACAATAAAGCGTTTCACGCA1200              TTAGAGCTTCCTTTTGTCTTTGGAAATCTGGACGGGTTGGAACGAATGGCAAAAGCGGAG1260              ATTACGGATGAGGTGAAACAGCTTTCTCACACGATACAATCCGCGTGGATCACGTTCGCT1320              AAAACAGGAAACCCAAGCACCGAAGCTGTGAATTGGCCGGCGTATCATGAAGAAACGAGA1380              GAGACGGTGATTTTAGACTCAGAGATTACGATCGAAAACGATCCCGAATCTGAAAAAAGG1440              CAGAAGCTATTCCCTTCAAAAGGAGAATAA1470                                            (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 489 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: enzyme                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      MetThrHisGlnIleValThrThrGlnTyrGlyLysValLysGlyThr                              151015                                                                        ThrGluAsnGlyValHisLeuTrpLysGlyIleProTyrAlaLysPro                              202530                                                                        ProValGlyGlnTrpArgPheLysAlaProGluProProGluValTrp                              354045                                                                        GluAspValLeuAspAlaThrAlaTyrGlyProIleCysProGlnPro                              505560                                                                        SerAspLeuLeuSerLeuSerTyrThrGluLeuProArgGlnSerGlu                              65707580                                                                      AspCysLeuTyrValAsnValPheAlaProAspThrProSerGlnAsn                              859095                                                                        LeuProValMetValTrpIleHisGlyGlyAlaPheTyrLeuGlyAla                              100105110                                                                     GlySerGluProLeuTyrAspGlySerLysLeuAlaAlaGlnGlyGlu                              115120125                                                                     ValIleValValThrLeuAsnTyrArgLeuGlyProPheGlyPheMet                              130135140                                                                     HisLeuSerSerPheAspGluAlaTyrSerAspAsnLeuGlyLeuLeu                              145150155160                                                                  AspGlnAlaAlaAlaLeuLysTrpValArgGluAsnIleSerAlaPhe                              165170175                                                                     GlyGlyAspProAspAsnValThrValPheGlyGluSerAlaGlyGly                              180185190                                                                     MetSerIleAlaAlaLeuLeuAlaMetProAlaAlaLysGlyLeuPhe                              195200205                                                                     GlnLysAlaIleMetGluSerGlyAlaSerArgThrMetThrLysGlu                              210215220                                                                     GlnAlaAlaSerThrAlaAlaAlaPheLeuGlnValLeuGlyIleAsn                              225230235240                                                                  GluSerGlnLeuAspArgLeuHisThrValAlaAlaGluAspLeuLeu                              245250255                                                                     LysAlaAlaAspGlnLeuArgIleAlaGluArgGluAsnIlePheGln                              260265270                                                                     LeuPhePheGlnProAlaLeuAspProLysThrLeuProGluGluPro                              275280285                                                                     GluLysSerIleAlaGluGlyAlaAlaSerGlyIleProLeuLeuIle                              290295300                                                                     GlyThrThrArgAspGluGlyTyrLeuPhePheThrProAspSerAsp                              305310315320                                                                  ValArgSerGlnGluThrLeuAspAlaAlaLeuGluTyrSerLeuGly                              325330335                                                                     LysProLeuAlaGluLysAlaAlaAspLeuTyrProArgSerLeuGlu                              340345350                                                                     SerGlnIleHisMetValThrAspLeuLeuPheTrpArgProAlaVal                              355360365                                                                     AlaPheAlaSerAlaGlnSerHisTyrAlaProValTrpMetTyrArg                              370375380                                                                     PheAspTrpHisProGluLysProProTyrAsnLysAlaPheHisAla                              385390395400                                                                  LeuGluLeuProPheValPheGlyAsnLeuAspGlyLeuGluArgMet                              405410415                                                                     AlaLysAlaGluIleThrAspGluValLysGlnLeuSerHisThrIle                              420425430                                                                     GlnSerAlaTrpIleThrPheAlaLysThrGlyAsnProSerThrGlu                              435440445                                                                     AlaValAsnTrpProAlaTyrHisGluGluThrArgGluThrValIle                              450455460                                                                     LeuAspSerGluIleThrIleGluAsnAspProGluSerGluLysArg                              465470475480                                                                  GlnLysLeuPheProSerLysGlyGlu                                                   485                                                                           (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1470 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM:                                                                 (C) INDIVIDUAL ISOLATE: 2-13f3                                                (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: complement (1..1470)                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      ATGACTCATCAAATAGTAACGACTCAATACGGCAAAGTAAAAGGCACAACGGAAAACGGC60                GTACATAAGTGGAAAGGCATCCCTTATGCCAAGCCGCCTGTCGGACAATGGCGTTTTAAA120               GCACCTGAGCCGCCTGAAGTGTGGGAAGATGTCCTTGATGCCACAGCGTACGGTCCTATT180               TGCCCGCAGCCGTCTGATTTGCTCTCACTGTCGTATACAGAGCTGCCCCGCCAGTCCGAG240               GATTGCTTGTATGTCAATGTATTTGCGCCTGACACTCCAAGTCAAAGTCTTCCTGTCATG300               GTGTGGATTCACGGAGGCGCTTTTTATCTTGGAGCGGGCAGTGAGCCATTGTATGACGGA360               TCAAAACTTGCGGCACAGGGAGAAGTCATTGTCGTTACATTGAACTATCGGCTGGGGCCG420               TTTGGCTTTTTGCACTTGTCTTCGTTTGATGAGGCGTATTCCGATAACCTTGGGCTTTTA480               GACCAAGCCGCCGCGCTGAAATGGGTGCGGGAGAATATCTCAGCGTTTGGCGGTGATCCC540               GATAACGTAACAGTATTTGGAGAATCCGCCGGCGGCATGAGCATTGCCGCGCTGCTCGCT600               ATGCCTGCGGCAAAAGGCCTGTTCCAGAAAGCGATCATGGAAAGCGGCGCTTCCCGAACA660               ATGACAAAAGAACAAGCGGCAAGCACTGCGGCTGCCTTTTTACAGGTCCTTGGGATTAAT720               GAGAGCCAGCTGGACAGATTGCATACTGTAGCAGCGGAAGATTTGCTTAAAGCGGCCGAT780               CAGCTTCGGATTGCAGAAAAAGAAAATATCTTTCAGCTGTTCTTCCAGCCCGCCCTTGAT840               CCGAAAACGCTGCCTGAAGAACCAGAAAAATCGATCGCAGAAGGGGCTGCTTCCGGCATT900               CCGCTATTGATTGGAACAACCCGTGATGAAGGATATTTATTTTTCACCCCGGATTCAGAC960               GTTCGTTCTCAGGAAACGCTTGATGCAGCACTCGAGTATTTACTAGGGAAGCCGCTGGCA1020              GAGAAAGCTGCCGATTTGTATCCGCGTTCTCTGGAAAGCCAAATTCATATGGTGACTGAT1080              TTATTATTTTGGCGCCCTGCCGTCGCCTTTGCATCCGCGCAGTCTCATTACGCCCCTGTC1140              TGGATGTACCGGTTCGATTGGCACCCGGAGAAGCCGCCGTACAATAAAGCGTTTCACGCA1200              TTAGAGCTTCCTTTTGTCTTTGGGAATCTGGACGGATTGGAACGAATGGCAAAAGCGGAG1260              ATTACGGATGAGGTGAAACAGCTTTCTCACACGATACAGTCCGCGTGGATCACGTTCGCT1320              AAAACAGGAAACCCAAGCACCGAAGCTGTGAATTGGCCGGCGTATCATGAAGAAACGAGA1380              GAGACGGTGATTTTAGACTCAGAGATTACGATCGAAAACGATCCCGAATCTGAAAAAAGG1440              CAGAAGCTATTCCCTTCAAAAGGAGAATAA1470                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 489 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: enzyme                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      MetThrHisGlnIleValThrThrGlnTyrGlyLysValLysGlyThr                              151015                                                                        ThrGluAsnGlyValHisLeuTrpLysGlyIleProTyrAlaLysPro                              202530                                                                        ProValGlyGlnTrpArgPheLysAlaProGluProProGluValTrp                              354045                                                                        GluAspValLeuAspAlaThrAlaTyrGlyProIleCysProGlnPro                              505560                                                                        SerAspLeuLeuSerLeuSerTyrThrGluLeuProArgGlnSerGlu                              65707580                                                                      AspCysLeuTyrValAsnValPheAlaProAspThrProSerGlnSer                              859095                                                                        LeuProValMetValTrpIleHisGlyGlyAlaPheTyrLeuGlyAla                              100105110                                                                     GlySerGluProLeuTyrAspGlySerLysLeuAlaAlaGlnGlyGlu                              115120125                                                                     ValIleValValThrLeuAsnTyrArgLeuGlyProPheGlyPheLeu                              130135140                                                                     HisLeuSerSerPheAspGluAlaTyrSerAspAsnLeuGlyLeuLeu                              145150155160                                                                  AspGlnAlaAlaAlaLeuLysTrpValArgGluAsnIleSerAlaPhe                              165170175                                                                     GlyGlyAspProAspAsnValThrValPheGlyGluSerAlaGlyGly                              180185190                                                                     MetSerIleAlaAlaLeuLeuAlaMetProAlaAlaLysGlyLeuPhe                              195200205                                                                     GlnLysAlaIleMetGluSerGlyAlaSerArgThrMetThrLysGlu                              210215220                                                                     GlnAlaAlaSerThrAlaAlaAlaPheLeuGlnValLeuGlyIleAsn                              225230235240                                                                  GluSerGlnLeuAspArgLeuHisThrValAlaAlaGluAspLeuLeu                              245250255                                                                     LysAlaAlaAspGlnLeuArgIleAlaGluLysGluAsnIlePheGln                              260265270                                                                     LeuPhePheGlnProAlaLeuAspProLysThrLeuProGluGluPro                              275280285                                                                     GluLysSerIleAlaGluGlyAlaAlaSerGlyIleProLeuLeuIle                              290295300                                                                     GlyThrThrArgAspGluGlyTyrLeuPhePheThrProAspSerAsp                              305310315320                                                                  ValArgSerGlnGluThrLeuAspAlaAlaLeuGluTyrLeuLeuGly                              325330335                                                                     LysProLeuAlaGluLysAlaAlaAspLeuTyrProArgSerLeuGlu                              340345350                                                                     SerGlnIleHisMetValThrAspLeuLeuPheTrpArgProAlaVal                              355360365                                                                     AlaPheAlaSerAlaGlnSerHisTyrAlaProValTrpMetTyrArg                              370375380                                                                     PheAspTrpHisProGluLysProProTyrAsnLysAlaPheHisAla                              385390395400                                                                  LeuGluLeuProPheValPheGlyAsnLeuAspGlyLeuGluArgMet                              405410415                                                                     AlaLysAlaGluIleThrAspGluValLysGlnLeuSerHisThrIle                              420425430                                                                     GlnSerAlaTrpIleThrPheAlaLysThrGlyAsnProSerThrGlu                              435440445                                                                     AlaValAsnTrpProAlaTyrHisGluGluThrArgGluThrValIle                              450455460                                                                     LeuAspSerGluIleThrIleGluAsnAspProGluSerGluLysArg                              465470475480                                                                  GlnLysLeuPheProSerLysGlyGlu                                                   485                                                                           (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1470 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM:                                                                 (C) INDIVIDUAL ISOLATE: 2-23e1                                                (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: complement (1..1470)                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      ATGACTCATCAAATAGTAACGACTCAATACGGCAAAGTAAAAGGCACAACGGAAAACGGC60                GTACATAAGTGGAAAGGCATCCCTTATGCCAAGCCGCCTGTCGGACAATGGCGTTTTAAA120               GCACCTGAGCCGCCTGAAGTGTGGGAAGATGTCCTTGATGCCACAGCGTACGGTCCTATT180               TGCCCGCAGCCGTCTGATTTGCTCTCACTGTCGTATACAGAGCTGCCCCGCCAGTCCGAG240               GATTGCTTGTATGTCAATGTATTTGCGCCTGACACTCCAAGTCAAAATCTTCCTGTCATG300               GTGTGGATTCACGGAGGCGCTTTTTATCTTGGAGCGGGCAGTGAGCCATTGTATGACGGA360               TCAAAACTTGCGGCACAGGGAGAAGTCATTGTCGTCACATTGAACTATCGGCTGGGGCCG420               TTTGGCTTTTTGCACTTGTCTTCGTTTGATGAGGCGTATTCCGATAACCTTGGGCTTTTA480               GACCAAGCCGCCGCGCTGAAATGGGTGCGGGAGAATATCTCAGCGTTTGGCGGTGATCCC540               GATAACGTAACAGTATTTGGAGAATCCGCCGGCGGCATGAGCATTGCCGCGCTGCTCGCT600               ATGCCTGCGGCAAAAGGCCTGTTCCAGAAAGCGATCATGGAAAGCGGCGCTTCCCGAACA660               ATGACAAAAGAACAAGCGGCAAGCACTGCGGCTGCCTTTTTACAGGTCCTTGGGATTAAT720               GAGAGCCAGCTGGACAGATTGCATACTGTAGCAGCGGAAGATTTGCTTAAAGCGGCCGAT780               CAGCTTCGGATTGCAGAAAAAGAAAATATCCTTCAGCTGTTCTTCCAGCCCGCCCTTGAT840               CCGAAAACGCTGCCTGAAGAACCAGAAAAATCGATCGCAGAAGGGGCTGCTTCCGGCATT900               CCGCTATTGATTGGAACAACCCGTGATGAAGGATATTTATTTTTCACCCCGGATTCAGAC960               GTTCGTTCTCAGGAAACGCTTGATGCAGCACTCGAGTATTTACTAGGGAAGCCGCTGGCA1020              GAGAAAGTTGCCGATTTGTATCCGCGTTCTCTGGAAAGCCAAATTCATATGATGACTGAT1080              TTATTATTTTGGCGCCCTGCCGTCGCCTTTGCATCCGCACAGTCTCATTACGCCCCTGTC1140              TGGATGTACCGGTTCGATTGGCACCCGGAGAAGCCGCCGTACAATAAAGCGTTTCACGCA1200              TTAGAGCTTCCTTTTGTCTTTGGAAATCTGGACGGATTGGAACGAATGGCAAAAGCGGAG1260              ATTACGGATGAGGTGAAACAGCTTTCTCACACGATACAATCCGCGTGGATCACGTTCGCT1320              AAAACAGGAAACCCAAGCACCGAAGCTGTGAATTGGCCGGCGTATCATGAAGAAACGAGA1380              GAGACGGTGATTTTAGACTCAGAGATTACGATCGAAAACGATCCCGAATCTGAAAAAAGG1440              CAGAAGCTATTCCCTTCAAAAGGAGAATAA1470                                            (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 489 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: enzyme                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      MetThrHisGlnIleValThrThrGlnTyrGlyLysValLysGlyThr                              151015                                                                        ThrGluAsnGlyValHisLeuTrpLysGlyIleProTyrAlaLysPro                              202530                                                                        ProValGlyGlnTrpArgPheLysAlaProGluProProGluValTrp                              354045                                                                        GluAspValLeuAspAlaThrAlaTyrGlyProIleCysProGlnPro                              505560                                                                        SerAspLeuLeuSerLeuSerTyrThrGluLeuProArgGlnSerGlu                              65707580                                                                      AspCysLeuTyrValAsnValPheAlaProAspThrProSerGlnAsn                              859095                                                                        LeuProValMetValTrpIleHisGlyGlyAlaPheTyrLeuGlyAla                              100105110                                                                     GlySerGluProLeuTyrAspGlySerLysLeuAlaAlaGlnGlyGlu                              115120125                                                                     ValIleValValThrLeuAsnTyrArgLeuGlyProPheGlyPheLeu                              130135140                                                                     HisLeuSerSerPheAspGluAlaTyrSerAspAsnLeuGlyLeuLeu                              145150155160                                                                  AspGlnAlaAlaAlaLeuLysTrpValArgGluAsnIleSerAlaPhe                              165170175                                                                     GlyGlyAspProAspAsnValThrValPheGlyGluSerAlaGlyGly                              180185190                                                                     MetSerIleAlaAlaLeuLeuAlaMetProAlaAlaLysGlyLeuPhe                              195200205                                                                     GlnLysAlaIleMetGluSerGlyAlaSerArgThrMetThrLysGlu                              210215220                                                                     GlnAlaAlaSerThrAlaAlaAlaPheLeuGlnValLeuGlyIleAsn                              225230235240                                                                  GluSerGlnLeuAspArgLeuHisThrValAlaAlaGluAspLeuLeu                              245250255                                                                     LysAlaAlaAspGlnLeuArgIleAlaGluLysGluAsnIleLeuGln                              260265270                                                                     LeuPhePheGlnProAlaLeuAspProLysThrLeuProGluGluPro                              275280285                                                                     GluLysSerIleAlaGluGlyAlaAlaSerGlyIleProLeuLeuIle                              290295300                                                                     GlyThrThrArgAspGluGlyTyrLeuPhePheThrProAspSerAsp                              305310315320                                                                  ValArgSerGlnGluThrLeuAspAlaAlaLeuGluTyrLeuLeuGly                              325330335                                                                     LysProLeuAlaGluLysValAlaAspLeuTyrProArgSerLeuGlu                              340345350                                                                     SerGlnIleHisMetMetThrAspLeuLeuPheTrpArgProAlaVal                              355360365                                                                     AlaPheAlaSerAlaGlnSerHisTyrAlaProValTrpMetTyrArg                              370375380                                                                     PheAspTrpHisProGluLysProProTyrAsnLysAlaPheHisAla                              385390395400                                                                  LeuGluLeuProPheValPheGlyAsnLeuAspGlyLeuGluArgMet                              405410415                                                                     AlaLysAlaGluIleThrAspGluValLysGlnLeuSerHisThrIle                              420425430                                                                     GlnSerAlaTrpIleThrPheAlaLysThrGlyAsnProSerThrGlu                              435440445                                                                     AlaValAsnTrpProAlaTyrHisGluGluThrArgGluThrValIle                              450455460                                                                     LeuAspSerGluIleThrIleGluAsnAspProGluSerGluLysArg                              465470475480                                                                  GlnLysLeuPheProSerLysGlyGlu                                                   485                                                                           (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1470 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM:                                                                 (C) INDIVIDUAL ISOLATE: 4-53d5                                                (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: complement (1..1470)                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      ATGACTCATCAAATAGTAACGACTCAATACGGCAAAGTAAAAGGCACAACGGAAAACGGC60                GTACATAAGTGGAAAGGCATCCCTTATGCCAAGCCGCCTGTCGGACAATGGCGTTTTAAA120               GCACCTGAGCCGCCTGAAGTGTGGGAAGATGTCCTTGATGCCACAGCGTACGGTCCTATT180               TGCCCGCAGCCGTCTGATTTGCTCTCACTGTCGTATACAGAGCTGCCCCGCCAGTCCGAG240               GATTGCTTGTACGTCAATGTATTTGCGCCTGACACTCCAAGTCAAAACCTTCCTGTCATG300               GTGTGGATTCACGGAGGCGCTTTTTATCTAGGAGCGGGCAGTGAGCCATTGTATGACGGA360               TCAAAACTTGCGGCACAGGGAGAAGTCATTGTCGTTACATTGAACTATCGGCTGGGGCCG420               TTTGGCTTTATGCACTTGTCTTCGTTTGATGAGGCGTATTCCGATAACCTTGGGCTTTTA480               GACCAAGCCGCCGCGCTGAAATGGGTGCGGGAGAATATCTCAGCGTTTGGCGGTGATCCC540               GATAACGTAACAGTATTTGGAGAATCCGCCGGCGGCATGAGCATTGCCGCGCTGCTCGCT600               ATGCCTGCGGCAAAAGGCCTGTTCCAGAAAGCGATCATGGAAAGCGGCGCTTCCCGAACA660               ATGACAAAAGAACAAGCGGCAAGCACTGCGGCTGCCTTTTTACAGGTCCTTGGGATCAAT720               GAGAGCCAGCTGGACAGATTGCATACTGTAGCAGCGGAAGATTTGCTTAAAGCGGCCGAT780               CAGCTTCGGATTGCAGAAAAAGAAAATATCTTTCAGCTGTTCTTCCAGCCCGCCCTTGAT840               CCGAAAACGCTGCCTGAAGAACCAGAAAAATCGATCGCAGAAGGGGCTGCTTCCGGCATT900               CCGCTATTGATTGGAACAACCCGTGATGAAGGATATTTATTTTTCACCCCGGATTCAGAC960               GTTCGTTCTCAGGAAACGCTTGATGCAGCACTCGAGTATTTACTAGGGAAGCCGCTGGCA1020              GAGAAAGTTGCCGATTTGTATCCGCGTTCTCTGGAAAGCCAAATTCATATGGTGACTGAT1080              TTATTATTTTGGCGCCCTGCCGTCGCCTTTGCATCCGCACAGTCTCATTACGCCCCTGTC1140              TGGATGTACCGGTTCGATTGGCACCCGGAGAAGCCGCCGTACAATAAAGCGTTTCACGCA1200              TTAGAGCTTCCTTTTGTCTTTGGAAATCTGGACGGGTTGGAACGAATGGCAAAAGCGGAG1260              ATTACGGATGAGGTGAAACAGCTTTCTCACACGATACAATCCGCGTGGATCACGTTCGCT1320              AAAACAGGAAACCCAAGCACCGAAGCTGTGAATTGGCCGGCGTATCATGAAGAAACGAGA1380              GAGACGGTGATTTTAGACTCAGAGATTACGATCGAAAACGATCCCGAATCTGAAAAAAGG1440              CAGAAGCTATTCCCTTCAAAAGGAGAATAA1470                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 489 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: enzyme                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      MetThrHisGlnIleValThrThrGlnTyrGlyLysValLysGlyThr                              151015                                                                        ThrGluAsnGlyValHisLeuTrpLysGlyIleProTyrAlaLysPro                              202530                                                                        ProValGlyGlnTrpArgPheLysAlaProGluProProGluValTrp                              354045                                                                        GluAspValLeuAspAlaThrAlaTyrGlyProIleCysProGlnPro                              505560                                                                        SerAspLeuLeuSerLeuSerTyrThrGluLeuProArgGlnSerGlu                              65707580                                                                      AspCysLeuTyrValAsnValPheAlaProAspThrProSerGlnAsn                              859095                                                                        LeuProValMetValTrpIleHisGlyGlyAlaPheTyrLeuGlyAla                              100105110                                                                     GlySerGluProLeuTyrAspGlySerLysLeuAlaAlaGlnGlyGlu                              115120125                                                                     ValIleValValThrLeuAsnTyrArgLeuGlyProPheGlyPheMet                              130135140                                                                     HisLeuSerSerPheAspGluAlaTyrSerAspAsnLeuGlyLeuLeu                              145150155160                                                                  AspGlnAlaAlaAlaLeuLysTrpValArgGluAsnIleSerAlaPhe                              165170175                                                                     GlyGlyAspProAspAsnValThrValPheGlyGluSerAlaGlyGly                              180185190                                                                     MetSerIleAlaAlaLeuLeuAlaMetProAlaAlaLysGlyLeuPhe                              195200205                                                                     GlnLysAlaIleMetGluSerGlyAlaSerArgThrMetThrLysGlu                              210215220                                                                     GlnAlaAlaSerThrAlaAlaAlaPheLeuGlnValLeuGlyIleAsn                              225230235240                                                                  GluSerGlnLeuAspArgLeuHisThrValAlaAlaGluAspLeuLeu                              245250255                                                                     LysAlaAlaAspGlnLeuArgIleAlaGluLysGluAsnIlePheGln                              260265270                                                                     LeuPhePheGlnProAlaLeuAspProLysThrLeuProGluGluPro                              275280285                                                                     GluLysSerIleAlaGluGlyAlaAlaSerGlyIleProLeuLeuIle                              290295300                                                                     GlyThrThrArgAspGluGlyTyrLeuPhePheThrProAspSerAsp                              305310315320                                                                  ValArgSerGlnGluThrLeuAspAlaAlaLeuGluTyrLeuLeuGly                              325330335                                                                     LysProLeuAlaGluLysValAlaAspLeuTyrProArgSerLeuGlu                              340345350                                                                     SerGlnIleHisMetValThrAspLeuLeuPheTrpArgProAlaVal                              355360365                                                                     AlaPheAlaSerAlaGlnSerHisTyrAlaProValTrpMetTyrArg                              370375380                                                                     PheAspTrpHisProGluLysProProTyrAsnLysAlaPheHisAla                              385390395400                                                                  LeuGluLeuProPheValPheGlyAsnLeuAspGlyLeuGluArgMet                              405410415                                                                     AlaLysAlaGluIleThrAspGluValLysGlnLeuSerHisThrIle                              420425430                                                                     GlnSerAlaTrpIleThrPheAlaLysThrGlyAsnProSerThrGlu                              435440445                                                                     AlaValAsnTrpProAlaTyrHisGluGluThrArgGluThrValIle                              450455460                                                                     LeuAspSerGluIleThrIleGluAsnAspProGluSerGluLysArg                              465470475480                                                                  GlnLysLeuPheProSerLysGlyGlu                                                   485                                                                           (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1470 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM:                                                                 (C) INDIVIDUAL ISOLATE: 5-1a12                                                (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: complement (1..1470)                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      ATGACTCATCAAATAGTAACGACTCAATACGGCAAAGTAAAAGGCACAACGGAAAACGGC60                GTACATAAGTGGAAAGGCATCCCTTATGCCAAGCCGCCTGTCGGACAATGGCGTTTTAAA120               GCACCTGAGCCGCCTGAAGTGTGGGAAGATGTCCTTGATGCCACAGCGTACGGTCCTGTT180               TGCCCGCAGCCGTCTGATTTGCTCTCACTGTCGTATACAGAGCTGCCCCGCCAGTCCGAG240               GATTGCTTGTATGTCAATGTATTTGCGCCTGACACTCCAAGTCAAAACCTTCCTGTCATG300               GTGTGGATTCACGGAGGCGCTTTTTATCTAGGAGCGGGCAGTGAGCCATTGTATGACGGA360               TCAAAACTTGCGGCACAGGGAGAAGTCATTGTCGTTACATTGAACTATCGGCTGGGGCCG420               TTTGGCTTTATGCACTTGTCTTCGTTTGATGAGGCGTATTCCGATAACCTTGGGCTTTTA480               GACCAAGCCGCCGCGCTGAAATGGGTGCGGGAGAATATCTCAGCGTTTGGCGGTGATCCC540               GATAACGTAACAGTATTTGGAGAATCCGCCGGCGGCATGAGCATTGCCGCGCTGCTCGCT600               ATGCCTGCGGCAAAAGGCCTGTTCCAGAAAGCGATCATGGAAAGCGGCGCTTCCCGAACA660               ATGACAAAAGAACAAGCGGCAAGCACTGCGGCTGCCTTTTTACAGGTCCTTGGGATTAAT720               GAGAGCCAGCTGGACAGATTGCATACTGTAGCAGCGGAAGATTTGCTTAAAGCGGCCGAT780               CAGCTTCGGATTGCAGAAAAAGAAAATATCTTTCAGCTGTTCTTCCAGCCCGCCCTTGAT840               CCGAAAACGCTGCCTGAAGAACCAGAAAAATCGATCGCAGAAGGGGCTGCTTCCGGCATT900               CCGCTATTGATTGGAACAACCCGTGATGAAGGATATTTATTTTTCACCCCGGATTCAGAC960               GTTCGTTCTCAGGAAACGCTTGATGCAGCACTCGAGTATTTACTAGGGAAGCCGCTGGCA1020              GAGAAAGCTGCCGATTTGTATCCGCGTTCTCTGGAAAGCCAAATTCATATGGTGACTGAT1080              TTATTATTTTGGCGCCCTGCCGTCGCCTTTGCATCCGCACAGTCTCATTACGCCCCTGTC1140              TGGATGTACCGGTTCGATTGGCACCCGGAGAAGCCGCCGTACAATAAAGCGTTTCACGCA1200              TTAGAGCTTCCTTTTGTCTTTGGAAATCTGGACGGGTTGGAACGAATGGCAAAAGCGGAG1260              ATTACGGATGAGGTGAAACAGCTTTCTCACACGATACAATCCGCGTGGATCACGTTCGCT1320              AAAACAGGAAACCCAAGCACCGAAGCTGTGAATTGGCCGGCGTATCATGAAGAAACGAGA1380              GAGACGGTGATTTTAGACTCAGAGATTACGATCGAAAACGATCCCGAATCTGAAAAAAGG1440              CAGAAGCTATTCCCTTCAAAAGGAGAATAA1470                                            (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 489 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: enzyme                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      MetThrHisGlnIleValThrThrGlnTyrGlyLysValLysGlyThr                              151015                                                                        ThrGluAsnGlyValHisLeuTrpLysGlyIleProTyrAlaLysPro                              202530                                                                        ProValGlyGlnTrpArgPheLysAlaProGluProProGluValTrp                              354045                                                                        GluAspValLeuAspAlaThrAlaTyrGlyProValCysProGlnPro                              505560                                                                        SerAspLeuLeuSerLeuSerTyrThrGluLeuProArgGlnSerGlu                              65707580                                                                      AspCysLeuTyrValAsnValPheAlaProAspThrProSerGlnAsn                              859095                                                                        LeuProValMetValTrpIleHisGlyGlyAlaPheTyrLeuGlyAla                              100105110                                                                     GlySerGluProLeuTyrAspGlySerLysLeuAlaAlaGlnGlyGlu                              115120125                                                                     ValIleValValThrLeuAsnTyrArgLeuGlyProPheGlyPheMet                              130135140                                                                     HisLeuSerSerPheAspGluAlaTyrSerAspAsnLeuGlyLeuLeu                              145150155160                                                                  AspGlnAlaAlaAlaLeuLysTrpValArgGluAsnIleSerAlaPhe                              165170175                                                                     GlyGlyAspProAspAsnValThrValPheGlyGluSerAlaGlyGly                              180185190                                                                     MetSerIleAlaAlaLeuLeuAlaMetProAlaAlaLysGlyLeuPhe                              195200205                                                                     GlnLysAlaIleMetGluSerGlyAlaSerArgThrMetThrLysGlu                              210215220                                                                     GlnAlaAlaSerThrAlaAlaAlaPheLeuGlnValLeuGlyIleAsn                              225230235240                                                                  GluSerGlnLeuAspArgLeuHisThrValAlaAlaGluAspLeuLeu                              245250255                                                                     LysAlaAlaAspGlnLeuArgIleAlaGluLysGluAsnIlePheGln                              260265270                                                                     LeuPhePheGlnProAlaLeuAspProLysThrLeuProGluGluPro                              275280285                                                                     GluLysSerIleAlaGluGlyAlaAlaSerGlyIleProLeuLeuIle                              290295300                                                                     GlyThrThrArgAspGluGlyTyrLeuPhePheThrProAspSerAsp                              305310315320                                                                  ValArgSerGlnGluThrLeuAspAlaAlaLeuGluTyrSerLeuGly                              325330335                                                                     LysProLeuAlaGluLysAlaAlaAspLeuTyrProArgSerLeuGlu                              340345350                                                                     SerGlnIleHisMetValThrAspLeuLeuPheTrpArgProAlaVal                              355360365                                                                     AlaPheAlaSerAlaGlnSerHisTyrAlaProValTrpMetTyrArg                              370375380                                                                     PheAspTrpHisProGluLysProProTyrAsnLysAlaPheHisAla                              385390395400                                                                  LeuGluLeuProPheValPheGlyAsnLeuAspGlyLeuGluArgMet                              405410415                                                                     AlaLysAlaGluIleThrAspGluValLysGlnLeuSerHisThrIle                              420425430                                                                     GlnSerAlaTrpIleThrPheAlaLysThrGlyAsnProSerThrGlu                              435440445                                                                     AlaValAsnTrpProAlaTyrHisGluGluThrArgGluThrValIle                              450455460                                                                     LeuAspSerGluIleThrIleGluAsnAspProGluSerGluLysArg                              465470475480                                                                  GlnLysLeuPheProSerLysGlyGlu                                                   485                                                                           (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1470 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM:                                                                 (C) INDIVIDUAL ISOLATE: consensus                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: complement (1..1470)                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      ATGACTCATCAAATAGTAACGACTCAATACGGCAAAGTAAAAGGCACAACGGAAAACGGC60                GTACATAAGTGGAAAGGCATCCCNTATGCCAAGCCGCCNGTCGGACAATGGCGTTTTAAA120               GCACCTGAGCCGCCTGAAGTGTGGGAAGATGTCCTTGATGCCACAGCGTACGGTCCTNTT180               TGCCCGCAGCCGTCTGATTTGCTCTCACTGTCGTATACAGAGCTGCCCCGCCAGTCCGAG240               GATTGCTTGTANGTCAATGTATTTGCGCCTGACACTCCANGTCAAANNCTTCCTGTCATG300               GTGTGGATTCACGGAGGCGCTTTTTATCTNGGAGCGGGCAGTGAGCCATTGTATGACGGA360               TCAAAACTTGCGGCACAGGGAGAAGTCATTGTCGTNACATTGAACTATCGGCTGGGGCCG420               TTTGGCTTTNTGCACTTGTCTTCGTTTGATGAGGCGTATTCCGATAACCTTGGGCTTTTA480               GACCAAGCCGCCGCGCTGAAATGGGTGCGGGAGAATATCTCAGCGTTTGGCGGTGATCCC540               GATAACGTAACAGTATTTGGAGAATCCGCCGGCGGCATGAGCATTGCCGCGCTGCTCGCT600               ATGCCTGCGGCAAAAGGCCTGTTCCAGAAAGCGATCATGGAAAGCGGCGCTTCCCGAACA660               ATGACAAAAGAACAAGCGGCAAGCACTGCGGCTGCCTTTTTACAGGTCCTTGGGATNAAT720               GAGAGCCAGCTGGACAGATTGCATACTGTAGCAGCGGAAGATTTGCTTAAAGCGGCCGAT780               CAGCTTCGGATTGCAGAAAAAGAAAATATCNTTCAGCTGTTCTTCCAGCCCGCCCTTGAT840               CCGAAAACGCTGCCTGAAGAACCAGAAAAATCGATCGCAGAAGGGGCTGCTTCCGGCATT900               CCGCTATTGATTGGAACAACCCGTGATGAAGGATATTTATTTTTCACCCCGGATTCAGAC960               GTTCNTTCTCAGGAAACGCTTGATGCAGCACTCGAGTATTTACTAGGGAAGCCGCTGGCA1020              GAGAAAGNTGCCGATTTGTATCCGCGTTCTCTGGAAAGCCAAATTCATATGNTGACTGAT1080              TTATTATTTTGGCGCCCTGCCGTCGCCTNTGCATCCGCNCAGTCTCATTACGCCCCTGTC1140              TGGATGTACCGGTTCGATTGGCACCCGGAGAAGCCGCCGTACAATAAAGCGTTTCACGCA1200              TTAGAGCTTCCTTTTGTCTTTGGNAATCTGGACGGNTTGGAACGAATGGCAAAAGCGGAG1260              ATTACGGATGAGGTGAAACAGCTTTCTCACACGATACANTCCGCGTGGATCACGTTCGCT1320              AAAACAGGAAACCCAAGCACCGAAGCTGTGAATTGGCCGGCGTATCATGAAGAAACGAGA1380              GAGACGGTGATTTTAGACTCAGAGATTACGATCGAAAACGATCCCGAATCTGAAAAAAGG1440              CAGAAGCTATTCCCTTCAAAAGGAGAATAA1470                                            (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 489 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: enzyme                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      MetThrHisGlnIleValThrThrGlnTyrGlyLysValLysGlyThr                              151015                                                                        ThrGluAsnGlyValHisLeuTrpLysGlyIleProTyrAlaLysPro                              202530                                                                        ProValGlyGlnTrpArgPheLysAlaProGluProProGluValTrp                              354045                                                                        GluAspValLeuAspAlaThrAlaTyrGlyProXaaCysProGlnPro                              505560                                                                        SerAspLeuLeuSerLeuSerTyrThrGluLeuProArgGlnSerGlu                              65707580                                                                      AspCysLeuTyrValAsnValPheAlaProAspThrProXaaGlnXaa                              859095                                                                        LeuProValMetValTrpIleHisGlyGlyAlaPheTyrLeuGlyAla                              100105110                                                                     GlySerGluProLeuTyrAspGlySerLysLeuAlaAlaGlnGlyGlu                              115120125                                                                     ValIleValValThrLeuAsnTyrArgLeuGlyProPheGlyPheXaa                              130135140                                                                     HisLeuSerSerPheAspGluAlaTyrSerAspAsnLeuGlyLeuLeu                              145150155160                                                                  AspGlnAlaAlaAlaLeuLysTrpValArgGluAsnIleSerAlaPhe                              165170175                                                                     GlyGlyAspProAspAsnValThrValPheGlyGluSerAlaGlyGly                              180185190                                                                     MetSerIleAlaAlaLeuLeuAlaMetProAlaAlaLysGlyLeuPhe                              195200205                                                                     GlnLysAlaIleMetGluSerGlyAlaSerArgThrMetThrLysGlu                              210215220                                                                     GlnAlaAlaSerThrAlaAlaAlaPheLeuGlnValLeuGlyIleAsn                              225230235240                                                                  GluSerGlnLeuAspArgLeuHisThrValAlaAlaGluAspLeuLeu                              245250255                                                                     LysAlaAlaAspGlnLeuArgIleAlaGluXaaGluAsnIleXaaGln                              260265270                                                                     LeuPhePheGlnProAlaLeuAspProLysThrLeuProGluGluPro                              275280285                                                                     GluLysSerIleAlaGluGlyAlaAlaSerGlyIleProLeuLeuIle                              290295300                                                                     GlyThrThrArgAspGluGlyTyrLeuPhePheThrProAspSerAsp                              305310315320                                                                  ValXaaSerGlnGluThrLeuAspAlaAlaLeuGluTyrXaaLeuGly                              325330335                                                                     LysProLeuAlaGluLysXaaAlaAspLeuTyrProArgSerLeuGlu                              340345350                                                                     SerGlnIleHisMetXaaThrAspLeuLeuPheTrpArgProAlaVal                              355360365                                                                     AlaXaaAlaSerAlaGlnSerHisTyrAlaProValTrpMetTyrArg                              370375380                                                                     PheAspTrpHisProGluLysProProTyrAsnLysAlaPheHisAla                              385390395400                                                                  LeuGluLeuProPheValPheGlyAsnLeuAspGlyLeuGluArgMet                              405410415                                                                     AlaLysAlaGluIleThrAspGluValLysGlnLeuSerHisThrIle                              420425430                                                                     GlnSerAlaTrpIleThrPheAlaLysThrGlyAsnProSerThrGlu                              435440445                                                                     AlaValAsnTrpProAlaTyrHisGluGluThrArgGluThrValIle                              450455460                                                                     LeuAspSerGluIleThrIleGluAsnAspProGluSerGluLysArg                              465470475480                                                                  GlnLysLeuPheProSerLysGlyGlu                                                   485                                                                           __________________________________________________________________________

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9. You, L. and Arnold, F. H. (1995). Directed Evolution of Subtilisin Ein Bacillus Subtilis to Enhance Total Activity in AqueousDimethylformamide, Protein Engineering, in press.

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15. Pohlenz, H. D., Boidol, W., Schuttke, I., and Streber, W. R. (1992).Purification and properties of an arthrobacter-oxydans p52 carbamatehydrolase specific for the herbicide phenmedipham andnucleotide-sequence of the corresponding gene. J. Bact. 174:6600-6607.

16. Tamura, N., Matsushita, Y., Iwama, T., Harada, S., Kishimoto, S. andItoh, K. (1991), Synthesis and biological activity of(S)-2-amino-3-(2,5-dihydro-5-oxo-4-isoxazolyl)propanoic acid (TAN-950 A)derivatives, Chem. Pharm. Bull. 39:1199-212.

17. Garg, H. G. and Jeanloz, R. W. (1974), Synthesis of protectedglycopeptides containing the amino acid sequences 34-37 and 34-38 ofbovine ribonuclease B, Carbohyd. Res. 32:37-46.

18. Egan, L. P. (1972), Synthesis and acid-catalyzed hydrolysis of3-0-glycosyl-L-serine and threonine, Carbohyd. Res. 23:261-73.

19. Hirayama, C., Ihara, H. and Shiraga, R. (1991), Induction ofleft-handed helical arrangement of 4-nitrobenzyl ester residues in castfilm from poly(L-glutamate) by using a cationic bilayer formingcompound, Chem. Lett. 8:1369-72.

20. Abiko, T. and Sekino, H. (1990), Synthesis of a thymosin β₄ -likepeptide, thymosin β₉ ^(Met), and its effect on low E-rosette-forminglymphocytes of lupus nephritis patients, Chem. Pharm. Bull. 38:2301-4.

21. Suzuki, K. and Endo, N. (1978), The β-p-nitrobenzyl ester tominimize side reaction during treatment of aspartyl peptides withmethanesulfonic acid, Chem. Pharm. Bull. 26:2269-74.

22. Barrett, G. C., Hardy, P. M., Harrow, T. A. and Rydon, H. N. (1972),Polypeptides. XXII. Synthesis of peptides of α-benzylphenylalanine bythe dicyclohexycarbodiimide method. J. Chem. Soc., Perkin Trans. 120:2634-8.

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What is claimed is:
 1. A modified para-nitrobenzyl esterase havingimproved hydrolysis activity in aqueous and aqueous/organic mediarelative to unmodified para-nitrobenzyl esterase of SEQ. ID. NO: 2 fromBacillus subtilis, wherein said modified para-nitrobenzyl esteraseconsists of unmodified para-nitrobenzyl esterase which has been modifiedby an amino acid substitution at one or more amino acid positionsselected from the group consisting of amino acid position numbers 60,94, 96, 144, 267, 271, 322, 334, 343, 358 and
 370. 2. A modifiedpara-nitrobenzyl esterase according to claim 1 wherein said one or moreamino acid substitions are selected from the group consisting of I60V,S94G, N96S, L144M, K267R, F271L, H322R, L334V, L334S, A343V, M358V andY370F.
 3. A modified para-nitrobenzyl esterase according to claim 1wherein said para-nitrobenzyl esterase is modified by the amino acidsubstitutions H322R and Y370F.
 4. A modified para-nitrobenzyl esteraseaccording to claim 1 wherein said para-nitrobenzyl esterase is modifiedby the amino acid substitutions H322R, M358V and Y370F.
 5. A modifiedpara-nitrobenzyl esterase according to claim 1 wherein saidpara-nitrobenzyl esterase is modified by the amino acid substitutionsL144M, H322R, M358V and Y370F.
 6. A modified para-nitrobenzyl esteraseaccording to claim 1 wherein said para-nitrobenzyl esterase is modifiedby the amino acid substitutions I60V, L144M, H322R, M358V and Y370F. 7.A modified para-nitrobenzyl esterase according to claim 1 wherein saidpara-nitrobenzyl esterase is modified by the amino acid substitutionsS94G, L144M, H322R, L334V, M358V and Y370F.
 8. A modifiedpara-nitrobenzyl esterase according to claim 1 wherein saidpara-nitrobenzyl esterase is modified by the amino acid substitutionsL144M, K267R, H322R, L334S, M358V and Y370F.
 9. A modifiedpara-nitrobenzyl esterase according to claim 1 wherein saidpara-nitrobenzyl esterase is modified by the amino acid substitutionsL144M, H322R, A343V, M358V and Y370F.
 10. A modified para-nitrobenzylesterase according to claim 1 wherein said para-nitrobenzyl esterase ismodified by the amino acid substitutions I60V, L144M, H322R, L334S,M358V and Y370F.
 11. A modified para-nitrobenzyl esterase according toclaim 1 wherein said para-nitrobenzyl esterase is modified by the aminoacid substitutions N96S, H322R, M358V and Y370F.
 12. A modifiedpara-nitrobenzyl esterase according to claim 1 wherein saidpara-nitrobenzyl esterase is modified by the amino acid substitutionsH322R, Y370F, F271L, A343V.